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Ginger and Zingerone Ameliorate Lipopolysaccharide-Induced Acute Systemic Inflammation in Mice, Assessed by Nuclear Factor-#B Bioluminescent Imaging Chien-Yun Hsiang, Hui-Man Cheng, Hsin-Yi Lo, Chia-Cheng Li, Pei-Chi Chou, Yu-Chen Lee, and Tin-Yun Ho J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01801 • Publication Date (Web): 15 Jun 2015 Downloaded from http://pubs.acs.org on June 16, 2015

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Journal of Agricultural and Food Chemistry

Ginger and Zingerone Ameliorate Lipopolysaccharide-Induced Acute Systemic Inflammation in Mice, Assessed by Nuclear Factor-κB Bioluminescent Imaging

Chien-Yun Hsiang,a,1 Hui-Man Cheng,b,1 Hsin-Yi Lo,c Chia-Cheng Li,d Pei-Chi Chou,b Yu-Chen Lee,e Tin-Yun Ho*,c,g a

Department of Microbiology, China Medical University, Taichung 40402, Taiwan

b

c

Graduate Institute of Chinese Medicine, China Medical University, Taichung 40402, Taiwan

d

e

School of Chinese Medicine, China Medical University, Taichung 40402, Taiwan

Graduate Institute of Cancer Biology, China Medical University, Taichung 40402, Taiwan

Graduate Institute of Acupuncture Science, China Medical University, Taichung 40402, Taiwan

g

Department of Health and Nutrition Biotechnology, Asia University, Taichung 41354, Taiwan

*Corresponding author. Telephone: +886 4 22053366 ext. 3302. Fax: +886 4 22032295. E-mail: [email protected] 1

These authors equally contributed to this work.

Short title: ginger and zingerone ameliorate LPS-induced inflammation

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ACS Paragon Plus Environment


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ABSTRACT

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Ginger is a commonly used spice in cooking. In this study, we comprehensively evaluated

3

the anti-inflammatory activities of ginger and its component zingerone in lipopolysaccharide

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(LPS)-induced acute systemic inflammation in mice via nuclear factor-κB (NF-κB)

5

bioluminescent imaging. Ginger and zingerone significantly suppressed LPS-induced NF-κB

6

activities in cells in a dose-dependent manner, and the maximal inhibition (84.5±3.5% and

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96.2±0.6%) was observed at 100 µg/ml ginger and zingerone, respectively. Moreover, dietary

8

ginger and zingerone significantly reduced LPS-induced proinflammatory cytokine

9

production in sera by 62.9±18.2% and 81.3±6.2%, respectively, and NF-κB bioluminescent

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signals in whole body by 26.9±14.3% and 38.5±6.2%, respectively. In addition, ginger and

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zingerone suppressed LPS-induced NF-κB-driven luminescent intensities in most organs and

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the maximal inhibition by ginger and zingerone was observed in small intestine.

13

Immunohistochemical staining further showed that ginger and zingerone decreased

14

interleukin-1β (IL-1β)-, CD11b-, and p65-positive areas in jejunum. In conclusion, our

15

findings suggested that ginger and zingerone were likely to be broad-spectrum

16

anti-inflammatory agents in most organs that suppressed the activation of NF-κB, the

17

production of IL-1β, and the infiltration of inflammatory cells in mice.

18 19

Key words: Ginger, zingerone, nuclear factor-κB, inflammation, bioluminescent imaging

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INTRODUCTION

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Ginger, the rhizome of Zingiber officinale, is one of the most commonly used spices in

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cooking. It is also a frequently used herb in alternative medicines. Clinical studies have

23

shown that ginger is effective in ameliorating nausea and vomiting caused by anti-retroviral

24

therapy, pregnancy, post-operation, and chemotherapy.1,2 It has add-on effects on reducing

25

knee pain and improving knee function in patients with symptomatic knee osteoarthritis.3,4 It

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is effective on pain relief in primary dysmenorrhea, eccentric exercise, and migraine.5,6

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Moreover, consumption of ginger is useful for patients with type 2 diabetes due to the

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reduction of glycated hemoglobin and the improvement of insulin resistance.7 These clinical

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data indicate the pharmacological application of ginger in medicine.

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Anti-inflammatory activities of ginger and its ingredients have been suggested in in vitro

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studies. For example, ginger extract inhibits the production of nitric oxide (NO) and

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proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated microglial cells, inhibits

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the activation of macrophages, and reduces the production of LPS-induced proinflammatory

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chemokines in bronchial epithelial cells.8,9 Moreover, ginger constituents, such as 6-shogaol,

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gingerol and 6-dehydroginerdione, display anti-inflammatory potentials in LPS-induced

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microglial cells or macrophages by inhibiting the production of cytokines.10,11

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Anti-inflammatory effects of ginger and it ingredients have also been evaluated in individual

38

organs, such as liver, brain, lung, and colon.12 For example, 6-shogaol suppresses the

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microglial activation in an in vivo neuroinflammatory model and shows a neuroprotective

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effect in transient global ischemia.13,14 Zingerone, a phenolic alkanone of ginger extract,

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attenuates LPS-induced acute lung injury and hepatic injury in mice.15 Moreover, zingerone

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improves experimental colitis in mice via nuclear factor-κB (NF-κB) activity in our previous

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study.16 However, these studies raise a question: do ginger extract and its constituents exhibit

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broad-spectrum anti-inflammatory effects in most organs?

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To address this question, we applied bioluminescent imaging on LPS-induced transgenic

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mice, which carried NF-κB-driven luciferase genes, to comprehensively monitor the

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anti-inflammatory effects of ginger and zingerone in whole body and organs. NF-κB

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bioluminescent imaging has been applied to assess host responses to the implantation of

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biomaterials and the exposure of ionizing radiation.17,18 It has been used to evaluate the

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anti-inflammatory potentials of vanillin and ginger extract on experimental colitis.16 It also

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has been utilized to monitor the anti-inflammatory effects of medicinal herbs on LPS-induced

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acute systemic inflammation and carbon tetrachloride-induced chronic hepatitis.19,20

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Immunohistochemical

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anti-inflammatory mechanisms of ginger and zingerone. Our findings suggested that ginger

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and zingerone were likely to be broad-spectrum anti-inflammatory agents in most organs that

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suppressed the activation of NF-κB, the production of interleukin-1β (IL-1β), and the

57

infiltration of inflammatory cells.

(IHC)

staining

was

further

performed

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to

elucidate

the

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MATERIALS AND METHODS Chemicals.

LPS

(from

Escherichia

coli

055:B5),

zingerone,

and

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3-(4,5-dimethylthiazol-2-yl-2,5-diohenyl tetrazolium bromide (MTT) were purchased from

62

Sigma (St. Louis, MO). MG-132, a NF-κB inhibitor, was purchased from Santa Cruz (Dallas,

63

TX). D-Luciferin was purchased from Xenogen (Hopkinton, MA). Mouse monoclonal

64

antibody against p65 was purchased from Chemicon (Temecula, CA). Rabbit polyclonal

65

antibodies against IL-1β and CD11b were purchased from Santa Cruz (Dallas, TX) and

66

Abcam (Cambridge, UK), respectively.

67 68

Preparation of Ginger Extract. Dried ginger was purchased from Xin Lung Chinese

69

Herbal Medicine Pharmacy (Taichung, Taiwan). The voucher specimen has been deposited in

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Graduate Institute of Chinese Medicine, China Medical University. Ginger was ground to a

71

fine powder and extracted by mixing 20 g powder with 100 ml ethanol at room temperature

72

with shaking. Three days later, the supernatant was collected and stored at -30°C for further

73

analysis.

74 75

Cell Culture. Recombinant HepG2/NF-κB cells, which carried NF-κB-driven luciferase

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genes, were constructed previously.19 HepG2/NF-κB cells were maintained in Dulbecco's

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modified Eagle's medium (Life Technologies, Gaithersburg, MD) supplemented with 10%

78

fetal bovine serum (Hyclone, Logan, Utah) and incubated at 37°C with 5% CO2.

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Luciferase Assay and Cell Viability Assay. HepG2/NF-κB cells (2×107 cells) were

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seeded in a 96-well plate and incubated at 37°C overnight. LPS (100 ng/ml), MG-132 (5 µM),

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or various amounts of ginger and zingerone were then added to cells and incubated at 37°C

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for 24 h. Cell viability was analyzed by MTT colorimetric assay as described previously.19

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Luciferase assay was performed as described previously.19 Relative NF-κB activity was

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calculated by dividing the relative luciferase unit (RLU) of compound-treated cells by the

86

RLU of solvent-treated cells.

87 88

Animal Experiments. Transgenic mice, carrying the luciferase genes driven by

89

NF-κB-responsive elements, were constructed as described previously.17 Mouse experiments

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were conducted under ethics approval from China Medical University Animal Care and Use

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Committee (Permit No. 97-28-N).

92

Six-week-old female transgenic mice were randomly divided into four groups of five mice:

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(1) mock, no treatment; (2) LPS, (3) LPS/ginger, and (4) LPS/zingerone. Mice were

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challenged intraperitoneally with 1 mg/kg LPS and then orally with 100 mg/kg ginger extract

95

or zingerone 10 min later. Four hours later, mice were imaged for the luciferase activity and

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subsequently sacrificed for ex vivo imaging and IHC staining.

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In Vivo and Ex Vivo Bioluminescence Imaging. Bioluminescence imaging was

99

performed as described previously.17 Briefly, for in vivo imaging, mice were injected

100

intraperitoneally with 150 mg/kg D-luciferin, placed in the IVIS Imaging System® 200 Series

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chamber (Xenogen, Hopkinton, MA) 5 min later, and imaged for 1 min. Photons emitted

102

from bodies were quantified using Living Image® software (Xenogen, Hopkinton, MA).The

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intensity of the signal from bodies was quantified as the sum of all photon counts per second

104

and presented as photon/sec. For ex vivo imaging, mice were injected with D-luciferin and

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sacrificed 5 min later. The organs were removed immediately, placed in the IVIS chamber,

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and imaged for 1 min. The intensity of signal was quantified as the sum of all detected photon

107

counts per second with the region of interest and presented as photon/sec/cm2/steradian (sr).

108 109

Cytokine Enzyme-Linked Immunosorbent Assay (ELISA). The amounts of

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proinflammatory cytokines, including IL-1β and tumor necrosis factor-α (TNF-α), in sera

111

were quantified using Quantikine® Mouse ELISA kits (R&D Systems, Minneapolis, MN).

112

Briefly, mouse sera were added to wells coated with anti-IL-1β or anti-TNF-α antibodies and

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incubated at room temperature for 2 h. Biotinylated anti-mouse IL-1β or TNF-α antibodies,

114

and avidin-horseradish peroxidase were added sequentially to wells. After a final wash,

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115

chromogenic substrate (tetramethylbenzidine) was added and the reaction was stopped with 2

116

N H2SO4. The absorbance at 450 nm was measured using an ELISA reader (Multiskan GO,

117

Thermo Scientific, Waltham, MA).

118 119

IHC Staining. Parafilm-embedded small intestines were cut into 5-µm-thick sections,

120

deparaffinized in xylene, and rehydrated in graded ethanol. Sections were incubated with

121

anti-p65, anti-IL-1β, or anti-CD11b antibodies overnight at 4°C and then incubated with a

122

biotinylated secondary antibody (Zymed Laboratories, Carlsbad, CA) for 20 min at room

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temperature. Finally, the sections were incubated with avidin-biotin complex reagent and

124

stained with 3,3'-diaminobenzidine according to manufacturer’s protocol (Histostain®-Plus,

125

Zymed Laboratories, Carlsbad, CA).

126 127

Statistics Analysis. Data were presented as mean ± standard error. Student’s t-test was

128

used for the comparison between two experiments. A value of p < 0.05 was considered

129

statistically significant.

130 131 132 133

RESULTS Ginger and Zingerone Suppressed LPS-Induced NF-κB Activities in Cells. Zingerone

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is a phenolic alkanone of ginger extract (Figure 1), and the content of zingerone was

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approximately 0.01 mg/ml in ethanolic extract of ginger by high-performance liquid

136

chromatography analysis (see Supplementary Figure 1 of the Supporting Information). We

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first analyzed the effects of ginger and zingerone on LPS-induced NF-κB activation in cells.

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HepG2/NF-κB cells were treated with LPS, followed by MG-132 or various amounts of

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ginger and zingerone. As shown in Figure 2, LPS increased the NF-κB activity by 4-fold,

140

compared with mock. MG-132, a well-known NF-κB inhibitor, significantly suppressed

141

LPS-induced NF-κB activities. Ginger and zingerone decreased NF-κB activities induced by

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LPS, and the decrease displayed a dose-dependent manner. The maximal inhibition

143

(84.5±3.5% and 96.2±0.6%) was observed at 100 µg/ml ginger and zingerone, respectively.

144

Moreover, zingerone was more effective than ginger on the inhibition of LPS-induced NF-κB

145

activity. No visible cytotoxic effects were observed, judged by MTT assay (data not shown).

146

These findings suggested that ginger and zingerone significantly suppressed NF-κB activities

147

induced by LPS in cells.

148 149

Ginger and Zingerone Suppressed LPS-Induced Inflammation in Mice. The in vivo

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anti-inflammatory effects of ginger and zingerone were then analyzed by NF-κB

151

bioluminescent imaging. Ginger extract or zingerone was orally given to transgenic mice,

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which has been challenged by 1 mg/kg LPS. The NF-κB-dependent bioluminescence was

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153

monitored 4 h later. As shown in Figure 3, LPS induced an approximately 4-fold increase in

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NF-κB-driven luminescent intensity, compared with mock. The induced luminescence was

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observed over the whole body, and the strongest luminescence appeared in the abdominal

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region. Ginger and zingerone significantly decreased the LPS-induced luminescent intensity

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by 26.9±14.3% and 38.5±6.2%, respectively. These findings suggested that ginger and

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zingerone suppressed LPS-induced NF-κB-dependent luminescence in mice.

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NF-κB plays a crucial role in the regulation of immunity. We wondered whether the

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intensity of NF-κB-driven luminescence was correlated with inflammation. The amount of

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proinflammatory cytokines, including IL-1β and TNF-α, in sera were therefore quantified by

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ELISA. As shown in Figure 4, LPS significantly increased the amount of IL-1β and TNF-α

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in sera by 55.6±7.6 and 172±57.5 fold, respectively. However, ginger and zingerone

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significantly decreased LPS-induced IL-1β and TNF-α production in sera. Ginger reduced the

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production of IL-1β and TNF-α by 73.5±23% and 63.9±18.2%, respectively, while zingerone

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decreased IL-1β and TNF-α production by 79.9±10.4% and 81.3±6.2%, respectively. These

167

data suggested that ginger and zingerone suppressed LPS-induced systemic inflammation in

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mice. Moreover, the correlation between NF-κB-dependent luminescent intensity and

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cytokine production indicated the representative of NF-κB-driven luminescence on the degree

170

of inflammation.

171

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Ginger and Zingerone Suppressed LPS-Induced Inflammation in Most Organs.

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Previous studies have shown that ginger or zingerone displayed anti-inflammatory efficacies

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in individual organs, such as liver, lung, brain, and colons. We would like to know whether

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ginger and zingerone exhibited broad-spectrum anti-inflammatory actions in most organs.

176

Transgenic mice were therefore challenged with LPS and orally administered with ginger

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extract and zingerone, and NF-κB-driven luminescence in organs was monitored 4 h later. As

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shown in Figure 5, luminescent intensities of organs were increased by LPS, suggesting that

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intraperitoneal injection of LPS induced inflammation in most organs. The maximal

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induction of luminescence by LPS was observed in kidney (15.9±8 fold), followed by

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intestine (15.2±6 fold), brain (10.4±1.4 fold), heart (5.7±1.5 fold), liver (4.7±1.9 fold), spleen

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(4.7±1.2 fold), lung (3.7±1.4 fold), and stomach (2.7±0.7 fold). Administration of ginger and

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zingerone significantly decreased LPS-induced NF-κB-driven luminescence in most organs.

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The maximal inhibition of LPS-induced luminescence by ginger and zingerone was observed

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in intestine, followed by kidney, liver, heart, and brain. Zingerone significantly decreased

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LPS-induced luminescence in lung, while ginger slightly decreased luminescent signals in

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lung. Moreover, both ginger and zingerone slightly decreased LPS-induced luminescent

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intensities in spleen.

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We further analyzed the anti-inflammatory effects of ginger and zingerone in different

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segments of small intestine. We divided the small intestine into 35 segments and the length

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ratio of duodenum, jejunum, and ileum was 1:3:2. The intensity of signal from each segment

192

was quantified as photon/sec. The suppression of LPS-induced luminescent signal by ginger

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or zingerone was further represented as the inhibitory percentage. As shown in Figure 6, LPS

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increased NF-κB-driven luminescence in whole small intestine, and the strong luminescence

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was observed from segment 17 to 30, which corresponded to the region between mid-jejunum

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and mid-ileum. Both ginger and zingerone suppressed LPS-induced bioluminescent intensity

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in whole small intestine, and the inhibition from segment 14 to 35 was > 50% by ginger and

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zingerone. Overall, these data suggested that ginger and zingerone displayed broad-spectrum

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anti-inflammatory activities in most organs. Additionally, ex vivo imaging first showed that

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LPS induced a more severe inflammation in the region spanning from mid-jejunum to

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mid-ileum. Moreover, LPS-induced luminescent signal in the junction of jejunum and ileum

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was suppressed efficiently by ginger and zingerone.

203 204

Ginger and Zingerone Inhibited LPS-Induced NF-κB Activation, IL-1β Production,

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and Inflammatory Cell Infiltration in Small Intestine. IHC staining was further performed

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to analyze the anti-inflammatory effects and mechanisms of ginger and zingerone in small

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intestine. As shown in Figure 7, the number of IL-1β-positive cells was increased by LPS,

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compared with mock. However, the expression of IL-1β-positive area was decreased by

209

ginger and zingerone. In addition, LPS increased the number of CD11b-positive cells,

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including monocytes and granulocytes, while ginger and zingerone inhibited the expression

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of CD11b-positive area. These data suggested that ginger and zingerone suppressed the

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production of IL-1β and the infiltration of inflammatory cells, resulting in the amelioration of

213

LPS-inflammation in small intestine.

214

Because NF-κB plays a critical role in inflammation, we analyzed the level of NF-κB

215

activity by IHC staining. The monoclonal antibody used here was against p65 nuclear

216

localization sequence, which was blocked by inhibitory IκB when NF-κB was inactivated.

217

LPS increased the number of p65-positive cells, while ginger and zingerone decreased the

218

p65-positive area. These findings suggested the inhibition of ginger and zingerone on

219

LPS-induced inflammation might be through NF-κB signaling pathway.

220 221 222

DISCUSSION

223

In this study, we comprehensively evaluated the anti-inflammatory effects of ginger and

224

zingerone by NF-κB bioluminescent imaging. Because of the light absorption by pigmented

225

molecules and the low spatial resolution of bioluminescent imaging, we performed ex vivo

226

imaging to monitor the effects of ginger and zingerone on individual organs. Previous studies

227

have shown that ginger extract and zingerone display anti-inflammatory activities in specific

228

organs. For example, ginger extracts ameliorate LPS-induced hepatic injury and experimental

13



229

colitis in mice.16,22 Administration of ginger extracts significantly represses paw and joint

230

swelling in rats with severe chronic adjuvant arthritis.23 Ginger also exhibits a protective role

231

on the diabetic brain by modulating the astroglial response to the injury in rats.24 Moreover,

232

zingerone attenuates LPS-induced lung injury in mice.15 By NF-κB bioluminescent imaging,

233

we found that administration of ginger and zingerone inhibited LPS-induced NF-κB-driven

234

luminescence in brain, lung, liver, and colon However, we newly identified that ginger and

235

zingerone rreduced LPS-induced luminescent intensities in heart, stomach, kidney, and small

236

intestine. In addition, the maximal inhibition of LPS-induced luminescent signal by ginger

237

and zingerone was observed in intestines, followed by kidney. The in vivo metabolism or

238

pharmacokinetics of ginger and zingerone has been reported. After oral administration of 2 g

239

ginger extract in human, the glucuronide and sulfate metabolites of ginger components, such

240

as gingerols and shogaol, are detected in plasma and gastrointestinal tract.25 Oral dosage (100

241

mg/kg) of zingerone in rats results in the urinary excretion of glucuronide and/or sulfate

242

conjugates of zingerone.26 The distribution of ginger extract and zingerone in gastrointestinal

243

tract and kidney might explain their anti-inflammatory effects in small intestine and kidney.

244

Because of the correlation between NF-κB-driven luminescent signals and inflammation, we

245

speculated that ginger and zingerone exhibited broad-spectrum anti-inflammatory activities

246

that suppressed the LPS-induced inflammation in various organs.

247

Anti-inflammatory mechanisms of ginger and its constituents have been analyzed in in

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vitro and in vivo studies. For instance, ginger extract ameliorates LPS-induced hepatic injury

249

via inhibiting the production of proinflammatory cytokines and attenuating the

250

mitogen-activated protein kinases and NF-κB signaling pathways.22 It also inhibits the

251

activities of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), and thus

252

inhibits the synthesis of prostaglandins and NO, mediators of inflammation.27 Ginger

253

derivatives, such as 6-shogaol, reduce osteoarthritis symptom by inhibiting toll-like receptor

254

4 (TLR-4)-mediated innate immunity and cathepsin-k activity.28 6-Shogaol also displays a

255

neuroprotective effect via inhibiting iNOS, COX-2, proinflammatory cytokines, and NF-κB

256

activities in LPS-treated microglial cells.14 In addition, 1-dehydro-10-gingerdione inhibits

257

TLR-4-mediated signaling cascades and cytokine expression via blockade of LPS binding to

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myeloid differentiation protein 2, a co-receptor of TLR-4 in macrophages.29 In this study, we

259

found that ginger and zingerone inhibited LPS-induced NF-κB activities in various organs in

260

mice. Ginger and zingerone also suppressed the nuclear translocation of NF-κB subunit p65,

261

the production of IL-1β, and the infiltration of granulocytes in small intestine. Overall, this

262

study suggested that ginger and zingerone shared a common anti-inflammatory mechanism

263

by inhibiting NF-κB activities and proinflammatory cytokine production in LPS-induced

264

systemic inflammation. Moreover, ginger exhibits a non-steroid anti-inflammatory activity

265

via inhibiting the activities of COX-2 and iNOS in other studies, probably explaining why

266

ginger displayed a broad-spectrum anti-inflammatory activity in various organs in this study.

15



267

Detailed anti-inflammatory effects of ginger and zingerone in small intestine were

268

evaluated here. It is interesting to find that LPS increased NF-κB-driven luminescence in the

269

entire small intestine, especially from the middle portion of jejunum to the end of ileum. It is

270

known that LPS activates macrophages, neutrophils, and dendritic cells via binding to TLR-4

271

and activating downstream NF-κB activity. The activated immune cells then initiate the

272

inflammatory response and present antigens to lymphocytes in lymph nodes.30 In comparison

273

with duodenum, jejunum and ileum have abundant Peyer's patches, organized lymphoid

274

nodules, in mice. Thus, we speculated that LPS induced maximal NF-κB-driven

275

luminescence in jejunum and ileum might result from the abundance of Peyer's patches. We

276

also found that ginger and zingerone suppressed LPS-induced NF-κB-dependent

277

bioluminescent signals in the entire small intestine, especially in the region between

278

mid-jejunum and mid-ileum. Zingerone possesses the vanillyl moiety, which is considered

279

important for the activation of vanilloid receptor 1 (VR1) expressed in nociceptive sensory

280

neurons.31 Recent study shows that activated VR1 protects against LPS-mediated renal injury

281

possibly via reducing renal inflammation responses.32 In addition, sensory VR1 has been

282

found to modulate cytokine response to LPS and thereby induce the subsequent

283

anti-inflammatory effect in the gut mucosa.33 VR1 nerve fibers are observed within enteric

284

ganglia of jejunum and ileum,34 probably explaining why zingerone displayed more activities

285

in jejunum and ileum than in duodenum.

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286

In conclusion, we comprehensively evaluated the anti-inflammatory effects of ginger and

287

zingerone on LPS-induced systemic inflammation via NF-κB bioluminescent imaging. Our

288

data showed for the first time that ginger and zingerone suppressed NF-κB-drive

289

luminescence in most organs. In addition, our findings suggested that ginger and zingerone

290

were likely to be broad-spectrum anti-inflammatory agents in most organs that suppressed the

291

activation of NF-κB, the production of IL-1β, and the infiltration of inflammatory cells.

292 293 294

ABBREVIATIONS USED

295

COX-2,

296

immunohistochemical; iNOS, inducible nitric oxide synthase; IL-1β, interleukin-1β; LPS,

297

lipopolysaccharide; MTT, 3-(4,5-dimethylthiazol-2-yl-2,5-diohenyl tetrazolium bromide; NO,

298

nitric oxide; NF-κB, nuclear factor-κB; RLU, relative luciferase unit; sr, steradian; TLR-4,

299

toll-like receptor 4; TNF-α, tumor necrosis factor-α; VR1, vanilloid receptor 1

cyclooxygenase-2;

ELISA,

enzyme-linked

immunosorbent

300 301

SUPPORTING INFORMATION

302

HPLC profile of ginger extract (Supplementary Figure S1).

303 304

FUNDING SOURCES

17


assay;

IHC,


Page 18 of 35

305

This work was supported by grants from Ministry of Science and Technology

306

(NSC101-2320-B-039-034-MY3,

NSC102-2632-B-039-001-MY3,

307

104-2815-C-039-012-B),

Medical

308

CMU103-SR-44), and CMU under the Aim for Top University Plan of the Ministry of

309

Education, Taiwan.

China

University

310 311

NOTES

312

The authors declare no competing financial interest.

313

18


and

(CMU102-NSC-04

MOST and

Page 19 of 35

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FIGURE CAPTIONS

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Figure 1. Ginger and zingerone. (A) Morphology of whole parts and cross sections of dried

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ginger. (B) Chemical structure of zingerone.

423 424

Figure 2. Effects of ginger and zingerone on LPS-induced NF-κB activities in cells.

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HepG2/NF-κB cells were treated with 100 ng/ml LPS and/or various amounts of ginger

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extract and zingerone. MG-132 (5 µM) was used as a positive control. Twenty-four hours

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later, NF-κB activity was measured by luciferase assay. Results are expressed as relative

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NF-κB activity, which is presented as the comparison with RLU relative to solvent-treated

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cells. Values are mean ± standard error (n=6). ###p < 0.001, compared with mock. **p

Ginger and Zingerone Ameliorate ... - ACS Publications

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