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Suppressive Effect of Ginsenoside Rg3 against LipopolysaccharideInduced Depression-Like Behavior and Neuroinflammation in Mice An Kang, Tong Xie, Dong Zhu, Jinjun Shan, liuqing di, and Xiao Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02386 • Publication Date (Web): 01 Aug 2017 Downloaded from http://pubs.acs.org on August 2, 2017

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Journal of Agricultural and Food Chemistry

Suppressive

Effect

of

Lipopolysaccharide-Induced

Ginsenoside Depression-Like

Rg3

against

Behavior and

Neuroinflammation in mice

An Kang †, §, Tong Xie †, Dong Zhu †, § , Jinjun Shan †, Liuqing Di *,†, §, Xiao Zheng *,‡



Jiangsu Key Laboratory of Pediatric Respiratory Disease and State Key Laboratory Cultivation

Base for TCM Quality and Efficacy, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China ‡

State Key Laboratory of Natural Medicines, School of Pharmacy, China Pharmaceutical

University, Nanjing 210009, China. §

Jiangsu Key Laboratory for Functional Substance of Chinese Medicine, Nanjing 210023, China.

Corresponding Authors: *(L.Q.D.) Phone: +86 25 86798226. Fax: +86 25 83271038. E-mail: [email protected]; *(X.Z.) Phone: +86 25 83271176 Fax: +86 25 83271176. E-mail: [email protected]. .

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Abstract

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Ginsenoside Rg3 (Rg3), a major active ingredient enriched in red ginseng, possesses

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well-confirmed immunoregulatory effects. Immune disturbance is a common trigger

4

and aggravating factor of depression. The aim of this study was to explore the effects

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of Rg3 on lipopolysaccharide (LPS)-induced depression-like behavior in mice and the

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involvement of immune regulation. Pretreatment with Rg3 (i.g., 20 and 40 mg/kg)

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effectively ameliorated LPS (i.p., 0.83 mg/kg) induced body weight loss, anorexia and

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immobility time in both the tail suspension test and the forced swimming test. Rg3

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attenuated the disturbed turnover of tryptophan and serotonin in the hippocampus

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brain, accompanied by decreased the mRNA expression of pro-inflammatory

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cytokines and indoleamine-2,3-dioxygenase (IDO). These central benefits were

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partially linked to the regulation of microglia activation and nuclear factor kappa B

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(NF-κB) pathway. In addition, Rg3 significantly reduced LPS-induced elevation of

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interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in plasma, and restored the

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systemic balance of tryptophan-kynurenine metabolism. Taken together, our results

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demonstrated that Rg3 was effective in ameliorating depressive-like behavior induced

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by immune activation, adding new evidence to support its health benefits by

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immunoregulation.

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ginsenoside

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Keywords:

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neuroinflammation, LPS

Rg3,

depressive-like

behavior,

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red

ginseng,

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INTRODUCTION

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Major depressive disorder (MDD) is a neuropsychiatric disease that has been

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predicted to become the second leading cause of disability worldwide1, 2. Although the

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complicated mechanism of MDD is yet to be fully understood, the important role of

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neuroimmune deregulation and inflammation has drawn much attention in

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accumulating clinical and preclinical studies2-4. Pro-inflammatory mediators were

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significantly elevated in various brain regions of MDD patients, which is most often

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accompanied by immune abnormality in periphery5, 6. These inflammatory changes

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have been well confirmed in animal models of depression, contributing to more global

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understandings of the neuroimmune basis of MDD7, 8. The causal role of immune

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disturbance in MDD is also supported by an increased likelihood to depressive

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symptoms in comorbid diseases such as inflammatory bowel disease, diabetes and

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cancer9. Mechanistically, it has been consistently suggested that sensing of

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inflammatory signals by the central nervous system (CNS), mainly through nervous

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system, humoral pathway or cytokine transport across the blood-brain barrier (BBB),

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may trigger microglia activation and perpetuated neuroinflammation3. Uncontrolled

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neuroimmune response is known to expedite an imbalanced action of neurotransmitter

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and neuroactive metabolites, which are intimately linked with deficits in neuronal

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function and behavioral control10. Studies in recent years have typically focused on

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the tryptophan-kynurenine (TRP-KYN) metabolic pathway, the over-activation of

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which is frequently observed in MDD patients and animal models of depression11.

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Increased KYN production, mediated by indoleamine-2,3-dioxygenase (IDO) due to 3

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inflammatory challenges12, 13, underlies the development of depressive-like behaviors

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partially via perivascular macrophage and microglia metabolism into neurotoxic

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quinolinic acid and 3-hydroxykynurenine11. Therefore, the metabolism of TRP to the

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neurotoxic KYN provides an important link between immune challenge,

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neuroinflammation and behavioral deficits.

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Ginseng or related products have been widely used as medical material and dietary

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supplements for various health benefits14-16. For some diseases or conditions,

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red ginseng possess superior effects than white/fresh ginseng, which is attributed to

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the fact that heating/steaming process produces large amount of bioactive compounds

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which are trace in fresh or white ginseng17. Ginsenoside Rg3 (Rg3, Figure 1A) is the

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major bioactive components enriched in red ginseng, which has been reported to

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possess numerous biological effects, including anti-oxidative, anti-inflammatory and

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immunomodulatory effects18-20. Although ginsenosides are well-known to regulate

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immune functions, the immunologic effect of individual component may differ for

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different diseases and immune parameters. Previous work in various animal studies

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has revealed that Rg3 exerted a fine control over immune responses in conditions such

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as lipopolysaccharide (LPS)-induced acute lung injury and lethal endotoxic shock21, 22,

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thus leading to reduced tissue injury and/or mortality from immune challenges.

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However, so far, there is limited information on the effects of Rg3 on

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immune-triggered behavioral deficits and neuroinflammatory parameters. Notably,

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although Rg3 has been very recently shown to have anti-inflammatory or 4

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anti-depressant effects in mice 23, 24, this was observed in models of chronic stress

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without direct pharmacological evidence for Rg3 on immune-triggered depressive

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behavior. In this study, a mice model of LPS-induced depression was employed to

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study the potential effects of Rg3 on immune-triggered depressive-like behavior.

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Furthermore, the association with central and systemic immunoregulation and

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TRP-KYN pathway metabolism were explored to gain some mechanistic insights.

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MATERIALS AND METHODS

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Drugs and reagents

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Rg3 (purity > 98%) was purchased from the College of Chemistry, Jilin University

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(Changchun, Jilin, China). LPS (E. Coli, 0127:B8), minocycline and standards of the

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TRP-KYN metabolites were purchased from Sigma-Aldrich Chemical Co. (St Louis,

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MO, USA). Anti-ionized calcium binding adaptor molecule 1 (Iba-1) antibody (Cat.

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No. 019-19741) for immunohistochemitry was purchased from Wako Pure Chemical

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Industries, Ltd. (Osaka, Japan). Antibodies for Western blotting analysis of nuclear

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factor kappa B (NF-κB) pathway (IκB-α, p-IκB-α, p-NF-κB p65, NF-κB p65) were

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provided by Cell Signaling Technology (Danvers, MA, USA). ELISA kits for the

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cytokines were purchased from BioLegend (San Diego, CA, USA). All the reagents

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and kits for the quantitative real-time PCR analysis were purchased from Takara Bio.

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Inc. (Takara, Dalian, Liaoning, China).

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Animals and treatment schedules 5

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ICR mice (male, 8 week old) were purchased from the Comparative Medicine Centre

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of Yangzhou University (Yangzhou, Jiangsu, China). Mice (3-4 per cage) were housed

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and maintained at controlled temperature (23 ± 1℃) and relative humidity (45–60%)

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under a normal 12 h light/dark cycle (lights on at 07:00 a.m.), with free access to food

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and water. Before use, mice were handled 2 min each day for 7 days to acclimate

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them to routine handing. All the animal studies were performed according to the US

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guidelines for the use and care of laboratory animals and approved by the Animal

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Ethics Committee of Nanjing University of Chinese medicine (SCXK2012-0004). All

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mice excluding the normal control group received a single challenge of LPS. LPS was

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freshly prepared in endotoxin-free saline and administered intraperitoneally (i.p.) at a

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dose of 0.83 mg/kg which proved reliable to induce depression-like behavior around

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24 h7,

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administered twice daily for 3 days prior to and on the same day of LPS injection at

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20 or 40 mg/kg. This dosing regimen were commonly adopted in the pharmacological

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study of Rg3 and some other ginsenosides in oral routes

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anti-inflammatory agent with confirmed anti-depressant effects 7, 27, was dissolved in

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0.9% NaCl containing 0.1% Tween 20 (Sigma-Aldrich) and then the pH of the

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minocycline was adjusted by lactic acid (Sigma-Aldrich) to 5.0. The minocyclin was

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i.p. administered at a dose of 50 mg/kg once daily for 2 days prior to and on the same

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day before LPS injection.

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Study design and sample collection

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One set of mice were used for the behavioral tests and then the hippocampus were

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. Rg3, suspended in 0.5% CMC-Na solution, was intragastrically (i.g.)

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19, 23, 26

. Minocycline, an

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collected for the analysis of serotonin, TRP and KYN metabolites (n=10). The plasma

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was also collected for the measurement of peripheral TRP-KYN metabolism. Food

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intake and body weight changes were also recorded at 22 h post LPS injection. In a

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subsequent study, mice were sacrificed at 24 h post LPS challenge and the

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hippocampuses were immediately dissected for the analysis of pro-inflammatory

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cytokines and IDO using quantitative real-time PCR (n=6). In the final study, mice

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were killed 4 h after LPS injection and plasma was collected and stored frozen (-80℃)

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until the analysis of pro-inflammatory cytokines (n=8). The hippocampuses were also

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collected for Western blotting analysis. The other mice were perfused with

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phosphate-buffered saline (PBS, pH 7.4) under anesthetization to eliminate blood

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components, and the brains were quickly collected from the skull and fixed in 4 %

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paraformaldehyde for 24 h for the immunohistochemical analysis.

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Behavioral tests

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Open field test (22 h post LPS injection), tail suspension (24 h post LPS injection) and

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forced swimming test (26 h post LPS injection) were employed for the behavior tests.

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The mice were brought to the behavioral testing room for at least one hour before the

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analysis. In order to assess possible effects of drug treatment on spontaneous

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locomotive activity, animals were subjected to the open-field test as previously

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described by us14. Briefly, mice were individually placed in the central square of a 60

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×60 cm gray wooden box with 50 cm high boundary walls and the floor was divided

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into 36 equal squares. Each mouse was video record for 5 min, and the number of

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crossing (the number of squares crossed) was calculated by two blinded observers. 7

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After each test, the box was cleansed with 5% ethanol.

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Forced swimming test (FST) was carried out as previously described with slight

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modification26. Briefly, mice were individually placed in an open cylinder (diameter

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15 cm, height 25 cm), containing 10 cm of water at 25 ± 1 ℃. Mice were gently

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placed onto the water and forced to swim for 6 min, and the total duration of

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immobility during the last 5 min was manually measured by two blinded observers.

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Mice were defined as immobility when they ceased struggling and remained floating

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motionless, only making those movements necessary to keep their head above the

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water. The water was changed between each testing session.

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Tail suspension test (TST) was conducted as previously described26. Briefly, mice

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were suspended by adhesive tape positioned about 1 cm from the tail tap, with the

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head 40 cm above the floor. The test was carried out for 6 min, and the duration of

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immobility was manually recorded during the final 5 min. Mice were considered

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immobile when they showed no signs of escape-oriented behavior.

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Determination of tryptophan metabolites

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Mouse hippocampus (about 20 mg) were weighted and sonicated in 100 µL of a 0.1

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M perchloric acid/10 µM ascorbate solution for 1 min, followed by centrifuge at

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12,000 g for 10 min at 4 ℃. An aliquot of 20 µL of supernatant was injected to a high

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performance liquid chromatography (HPLC) system equipped with a fluorescence

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detector (Waters, USA). For the determination of serotonin (5-HT) and its metabolite

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5-hydroxyindoleacetic acid (5-HIAA), samples were eluted with a mobile phase

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containing 25 mM acetate buffered with 0.75 mM sodium heptanesulfonate (pH 8

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3.9)-methanol (85:15, v/v). The excitation and emission wavelengths were set at 305

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and 360 nm, respectively. Samples were then subjected to a Waters 2695 HPLC

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system equipped with a UV detector (Waters Corp., Milford, MA) for TRP-KYN

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pathway analysis. Briefly, the separation was carried out on a Hypersil ODS column

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(250 mm * 4.6 mm, 5 µm), and the mobile consisted of 25 mM acetate (pH 5.0) and

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methanol (90: 10, v/v) at a flow rate of 1.0 mL/min. The concentration of 5-HT,

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5-HIAA, TRP and KYN were expressed as nanogram per gram of wet tissue weight.

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Enzyme-linked immunosorbent assay (ELISA)

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The concentration of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in

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plasma were measured using commercially available ELISA kits according to

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manufacturer’s instructions. Assays were sensitive to 7.8 pg/mL of IL-6 and TNF-α,

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and inter- and intra-assay coefficients of variation were less than 10%.

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Quantitative real-time PCR

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Total RNA from the hippocampus samples was extracted using Trizol reagent. RNA

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was reverse transcribed to cDNA using a Takara PrimeScript 1st Strand cDNA

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Synthesis Kit according to the manufacturer’s instructions. Real-Time PCR analysis

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was performed in the Applied Biosystems 7500 Fast Real-Time PCR System

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(Applied Biosystems Co., Carlsbad, California, USA). The sequences of primers

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used

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IL-1β,sense5’-CTGTGTCTTTCCCGTGGACC-3’;antisense5’-CAGCTCATATGGG

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TCCGACA-3’; IL-6, sense 5’- CCAGAAACCGCTATGAAGTTCCT-3’; antisense

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5’-

were

as

CACCAGCATCAGTCCCAAGA-3’;

TNF-α,

follows:

sense

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GTGGAACTG-3’; antisense 5’-CAGCTCATATGGGTCCGACA-3’; IDO, sense

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5’-GTACATCACCATGGCGTATG-3’;antisense5’-ACCGCCTGGAGTTCTGGAA-

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3’;β-actin,sense5’-TCTGGCACCACACCTTCTA-3’;antisense5’-AGGCATACAGG

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GACAGCA C-3’. The SYBR Green I PCR mix kit was used to quantify gene

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expression. LightCycler reactions were performed in a total volume of 20 µL based on

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the manufacturer’s instructions. Three replicates were performed for each quantitative

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PCR run. The mRNA concentrations of all target genes were normalized to that of the

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β-actin in each sample (using Delta-delta Ct method). Results are expressed as fold

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increases in mRNA compared to those in the hippocampus of control mice.

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Immunohistochemistry of Iba-1

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Microglia activation in brain tissue was observed using immunohistochemical

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analysis. Briefly, coronal sections of the brain with 50 µm thick were obtained using a

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Vibratome (Leica Microsystems, Bensheim, Germany). After washing twice in PBS

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(pH 7.4) for 10 min, endogenous peroxidase activity was quenched for 20min in 3 %

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hydrogen peroxide in methanol and washed twice in PBS for each 10 min later. Then,

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the brain sections were blocked in 5 % bovine serum albumin (BSA) solution for 1 h

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and incubated overnight at 4 ℃ with a primary rabbit polyclonal antibodies to Iba-1,

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a marker of microglia (diluted 1:500). After incubation with the primary antibody,

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sections were washed and incubated for 2 h at room temperature with the biotinylated

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goat anti-rabbit IgG (Santa Cruze Biotechnology, Inc., USA; diluted 1:200). After

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three washes in buffer, sections were revealed by chromogen DAB reaction for up to

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10 min. Finally, sections were dehydrated, mounted on gelatinized slides and 10

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examined with a light microscopy (Olympus, Tokyo, Japan). The number of Iba-1

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immunoreactive cells was estimated with the optical dissector method by two blinded

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observers. Three sections and four fields per section were chosen for the analysis in

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each mouse.

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Western blotting analysis

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The hippocampus sample for the western blot analysis was collected 4 h post LPS

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challenge and then stored at -80℃ until analysis. The hippocampus tissues were

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homogenized and lysed in the RIPA buffer supplemented with 1mM PMSF and 1%

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protease inhibitor cocktail (Beyotime Biotechnology, Nantong, Jiangsu, China) and

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then homogenates were centrifuged at 13000 g for 10 min at 4 °C to obtain the

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supernatants. The samples were separated on a sodium dodecyl sulfate

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polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF

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membrane (Bio-Rad, CA, USA). The membrane was blocked with 5% bovine serum

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albumin (BSA) in Tris-buffered Tween 20 (TBS/T, 0.1%) for 1 h at 37°C, followed by

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incubation with the primary antibodies to p-IκBα (Ser32), IκB-α, p-NF-κB p65

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(Ser536), NF-κB p65 overnight at 4°C. GAPDH, detected with 1:2000 diluted rabbit

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monoclonal primary antibody (Bioworld, MN, USA ) was used as the loading control.

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After that, the membrane was reacted with horserdish peroxide-conjugated anti-mouse

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IgG (1:5000) or anti-rabbit IgG (1:5000) secondary antibody solution for 1 h in

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TBS/T including 5% BSA. For chemiluminescence detection, the membranes were

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incubated by chemiluminescence with ECL reagent (Invitrogen, Carlsbad, CA, USA).

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The density of the band was measured by ImageJ software (National Institutes of 11

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Health, Bethesda, MD, USA).

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Statistical analysis

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Data were expressed as the mean ± standard deviation (SD). One-way analysis of

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variance (ANOVA) with Tukey multiple comparison test was used to determine

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potential significantly differences between control and experimental groups (SPSS,

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version 19.0, SPSS, Inc., Chicago, IL, USA). For all the analysis, a value of p < 0.05

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or p < 0.01 was considered statistically significant.

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RESULTS

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Rg3 ameliorated LPS-induced depressive-like behavior

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Typically, peripheral challenge of LPS triggers weight loss, anorexia and depressive

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symptoms within 24 h. As illustrated in Figure 1, food intake and body weight were

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significantly reduced in LPS-challenged mice as compared to control mice (p < 0.01)

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at 24 h post LPS challenge. By contrast, Rg3 (20, 40 mg/kg) or minocycline (50

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mg/kg) treatment conferred significant protective effects against LPS-associated

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weight loss (Figure 1B) and anorexia (Figure 1C) (p < 0.01). The potential

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alleviating effect of Rg3 on LPS-induced depression-like behavior in mice was then

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investigated.

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Open field test showed that neither LPS treatment nor drug administration (Rg3 or

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minocycline) produced any significant effects on the locomotor activity of mice at the

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time of behavioral tests (Figure 1D). Next, in the FST (Figure 1E), Rg3

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(103.58±16.82 for 20mg/kg and 86.86±18.22 for 40 mg/kg) or minocycline 12

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(57.48±11.29) significantly shortened the immobility time as compared with the

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LPS-treated group (128.23±18.78) (p ˂ 0.05 and p ˂ 0.01). Similar results could be

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seen in the TST that LPS challenge significantly increased the immobility time in TST

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when comparing to control mice (164.84±26.87 vs 65.87±11.51, p