Suppressive Effect of Ginsenoside Rg3 against Lipopolysaccharide

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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]. .

1

ACS Paragon Plus Environment


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Abstract

2

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

3

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

5

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

6

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

7

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

8

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

9

attenuated the disturbed turnover of tryptophan and serotonin in the hippocampus

10

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

11

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

12

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

13

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

14

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

15

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

16

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

17

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

18

immunoregulation.

19

ginsenoside

20

Keywords:

21

neuroinflammation, LPS

Rg3,

depressive-like

behavior,

22 23 24 25 26 27


red

ginseng,

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INTRODUCTION

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

30

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

31

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

32

neuroimmune deregulation and inflammation has drawn much attention in

33

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

34

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

35

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

36

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

37

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

38

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

39

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

40

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

41

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

42

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

43

may trigger microglia activation and perpetuated neuroinflammation3. Uncontrolled

44

neuroimmune response is known to expedite an imbalanced action of neurotransmitter

45

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

46

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

47

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

48

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

49

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



50

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

51

partially via perivascular macrophage and microglia metabolism into neurotoxic

52

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

53

neurotoxic KYN provides an important link between immune challenge,

54

neuroinflammation and behavioral deficits.

55 56

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

57

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

58

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

59

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

60

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

61

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

62

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

63

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

64

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

65

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

66

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

67

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

68

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

69

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

70

immune-triggered behavioral deficits and neuroinflammatory parameters. Notably,

71

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

73

without direct pharmacological evidence for Rg3 on immune-triggered depressive

74

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

75

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

76

Furthermore, the association with central and systemic immunoregulation and

77

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

78 79 80

MATERIALS AND METHODS

81

Drugs and reagents

82

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

83

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

84

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

85

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

86

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

87

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

88

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

89

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

90

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

91

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

92

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

93

Animals and treatment schedules 5



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

95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

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

103

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

104

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

105

24 h7,

106

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

107

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

108

study of Rg3 and some other ginsenosides in oral routes

109

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

110

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

111

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

112

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

113

day before LPS injection.

114

Study design and sample collection

115

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

25

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

6


19, 23, 26

. Minocycline, an

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116

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

117

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

118

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

119

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

120

hippocampuses were immediately dissected for the analysis of pro-inflammatory

121

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

122

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

123

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

124

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

125

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

126

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

127

paraformaldehyde for 24 h for the immunohistochemical analysis.

128

Behavioral tests

129

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

130

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

131

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

132

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

133

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

134

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

135

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

136

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

137

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



138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

water. The water was changed between each testing session.

147

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

148

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

149

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

150

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

151

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

152

Determination of tryptophan metabolites

153

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

154

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

155

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

156

performance liquid chromatography (HPLC) system equipped with a fluorescence

157

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

158

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

159

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

161

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

162

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

163

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

164

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

165

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

166

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

167

Enzyme-linked immunosorbent assay (ELISA)

168

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

169

plasma were measured using commercially available ELISA kits according to

170

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

171

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

172

Quantitative real-time PCR

173

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

174

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

175

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

176

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

177

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

178

used

179

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

180

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

181

5’-

were

as

CACCAGCATCAGTCCCAAGA-3’;

TNF-α,

follows:

sense

9


5’-ATCCGCGAC


182

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

183

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

184

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

185

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

186

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

187

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

188

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

189

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

190

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

191

Immunohistochemistry of Iba-1

192

Microglia activation in brain tissue was observed using immunohistochemical

193

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

194

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

195

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

196

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

197

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

198

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

199

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

200

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

201

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

202

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

203

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

205

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

206

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

207

each mouse.

208

Western blotting analysis

209

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

210

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

211

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

212

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

213

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

214

supernatants. The samples were separated on a sodium dodecyl sulfate

215

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

216

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

217

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

218

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

219

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

220

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

221

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

222

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

223

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

224

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

225

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


−


226

Health, Bethesda, MD, USA).

227

Statistical analysis

228

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

229

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

230

potential significantly differences between control and experimental groups (SPSS,

231

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

232

or p < 0.01 was considered statistically significant.

233 234

RESULTS

235

Rg3 ameliorated LPS-induced depressive-like behavior

236

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

237

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

238

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

239

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

240

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

241

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

242

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

243

investigated.

244

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

245

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

246

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

247

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


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248

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

249

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

250

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

251

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

Suppressive Effect of Ginsenoside Rg3 against Lipopolysaccharide

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