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
School of Chinese Medicine, China Medical University, Taichung 40402, Taiwan
Graduate Institute of Chinese Medicine, China Medical University, Taichung 40402, Taiwan
d
Graduate Institute of Cancer Biology, China Medical University, Taichung 40402, Taiwan
e
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: cyhsiang@mail.cmu.edu.tw
1
These authors equally contributed to this work.
Short title: ginger and zingerone ameliorate LPS-induced inflammation
1
1
ABSTRACT
2
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
4
(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
7
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
10
signals in whole body by 26.9±14.3% and 38.5±6.2%, respectively. In addition, ginger and
11
zingerone suppressed LPS-induced NF-κB-driven luminescent intensities in most organs and
12
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
2
20
INTRODUCTION
21
Ginger, the rhizome of Zingiber officinale, is one of the most commonly used spices in
22
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
26
is effective on pain relief in primary dysmenorrhea, eccentric exercise, and migraine.5,6
27
Moreover, consumption of ginger is useful for patients with type 2 diabetes due to the
28
reduction of glycated hemoglobin and the improvement of insulin resistance.7 These clinical
29
data indicate the pharmacological application of ginger in medicine.
30
Anti-inflammatory activities of ginger and its ingredients have been suggested in in vitro
31
studies. For example, ginger extract inhibits the production of nitric oxide (NO) and
32
proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated microglial cells, inhibits
33
the activation of macrophages, and reduces the production of LPS-induced proinflammatory
34
chemokines in bronchial epithelial cells.8,9 Moreover, ginger constituents, such as 6-shogaol,
35
gingerol and 6-dehydroginerdione, display anti-inflammatory potentials in LPS-induced
36
microglial cells or macrophages by inhibiting the production of cytokines.10,11
37
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
3
39
microglial activation in an in vivo neuroinflammatory model and shows a neuroprotective
40
effect in transient global ischemia.13,14 Zingerone, a phenolic alkanone of ginger extract,
41
attenuates LPS-induced acute lung injury and hepatic injury in mice.15 Moreover, zingerone
42
improves experimental colitis in mice via nuclear factor-κB (NF-κB) activity in our previous
43
study.16 However, these studies raise a question: do ginger extract and its constituents exhibit
44
broad-spectrum anti-inflammatory effects in most organs?
45
To address this question, we applied bioluminescent imaging on LPS-induced transgenic
46
mice, which carried NF-κB-driven luciferase genes, to comprehensively monitor the
47
anti-inflammatory effects of ginger and zingerone in whole body and organs. NF-κB
48
bioluminescent imaging has been applied to assess host responses to the implantation of
49
biomaterials and the exposure of ionizing radiation.17,18 It has been used to evaluate the
50
anti-inflammatory potentials of vanillin and ginger extract on experimental colitis.16 It also
51
has been utilized to monitor the anti-inflammatory effects of medicinal herbs on LPS-induced
52
acute systemic inflammation and carbon tetrachloride-induced chronic hepatitis.19,20
53
Immunohistochemical
54
anti-inflammatory mechanisms of ginger and zingerone. Our findings suggested that ginger
55
and zingerone were likely to be broad-spectrum anti-inflammatory agents in most organs that
56
suppressed the activation of NF-κB, the production of interleukin-1β (IL-1β), and the
57
infiltration of inflammatory cells.
(IHC)
staining
was
4
further
performed
to
elucidate
the
58
59
60
MATERIALS AND METHODS
Chemicals.
LPS
(from
Escherichia
coli
055:B5),
zingerone,
and
61
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
70
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
76
genes, were constructed previously.19 HepG2/NF-κB cells were maintained in Dulbecco's
5
77
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.
79
80
Luciferase Assay and Cell Viability Assay. HepG2/NF-κB cells (2×107 cells) were
81
seeded in a 96-well plate and incubated at 37C overnight. LPS (100 ng/ml), MG-132 (5 μM),
82
or various amounts of ginger and zingerone were then added to cells and incubated at 37C
83
for 24 h. Cell viability was analyzed by MTT colorimetric assay as described previously.19
84
Luciferase assay was performed as described previously.19 Relative NF-κB activity was
85
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-B-responsive elements, were constructed as described previously.17 Mouse experiments
90
were conducted under ethics approval from China Medical University Animal Care and Use
91
Committee (Permit No. 97-28-N).
92
Six-week-old female transgenic mice were randomly divided into four groups of five mice:
93
(1) mock, no treatment; (2) LPS, (3) LPS/ginger, and (4) LPS/zingerone. Mice were
94
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
6
96
subsequently sacrificed for ex vivo imaging and IHC staining.
97
98
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
101
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
103
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
105
sacrificed 5 min later. The organs were removed immediately, placed in the IVIS chamber,
106
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
110
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
113
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,
7
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
123
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
8
134
is a phenolic alkanone of ginger extract (Figure 1), and the content of zingerone was
135
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
137
first analyzed the effects of ginger and zingerone on LPS-induced NF-κB activation in cells.
138
HepG2/NF-κB cells were treated with LPS, followed by MG-132 or various amounts of
139
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
142
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
150
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,
152
which has been challenged by 1 mg/kg LPS. The NF-κB-dependent bioluminescence was
9
153
monitored 4 h later. As shown in Figure 3, LPS induced an approximately 4-fold increase in
154
NF-κB-driven luminescent intensity, compared with mock. The induced luminescence was
155
observed over the whole body, and the strongest luminescence appeared in the abdominal
156
region. Ginger and zingerone significantly decreased the LPS-induced luminescent intensity
157
by 26.9±14.3% and 38.5±6.2%, respectively. These findings suggested that ginger and
158
zingerone suppressed LPS-induced NF-κB-dependent luminescence in mice.
159
NF-κB plays a crucial role in the regulation of immunity. We wondered whether the
160
intensity of NF-κB-driven luminescence was correlated with inflammation. The amount of
161
proinflammatory cytokines, including IL-1β and TNF-α, in sera were therefore quantified by
162
ELISA. As shown in Figure 4, LPS significantly increased the amount of IL-1β and TNF-α
163
in sera by 55.6±7.6 and 172±57.5 fold, respectively. However, ginger and zingerone
164
significantly decreased LPS-induced IL-1β and TNF-α production in sera. Ginger reduced the
165
production of IL-1β and TNF-α by 73.5±23% and 63.9±18.2%, respectively, while zingerone
166
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
168
mice. Moreover, the correlation between NF-κB-dependent luminescent intensity and
169
cytokine production indicated the representative of NF-κB-driven luminescence on the degree
170
of inflammation.
171
10
172
Ginger and Zingerone Suppressed LPS-Induced Inflammation in Most Organs.
173
Previous studies have shown that ginger or zingerone displayed anti-inflammatory efficacies
174
in individual organs, such as liver, lung, brain, and colons. We would like to know whether
175
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
177
extract and zingerone, and NF-κB-driven luminescence in organs was monitored 4 h later. As
178
shown in Figure 5, luminescent intensities of organs were increased by LPS, suggesting that
179
intraperitoneal injection of LPS induced inflammation in most organs. The maximal
180
induction of luminescence by LPS was observed in kidney (15.9±8 fold), followed by
181
intestine (15.2±6 fold), brain (10.4±1.4 fold), heart (5.7±1.5 fold), liver (4.7±1.9 fold), spleen
182
(4.7±1.2 fold), lung (3.7±1.4 fold), and stomach (2.7±0.7 fold). Administration of ginger and
183
zingerone significantly decreased LPS-induced NF-κB-driven luminescence in most organs.
184
The maximal inhibition of LPS-induced luminescence by ginger and zingerone was observed
185
in intestine, followed by kidney, liver, heart, and brain. Zingerone significantly decreased
186
LPS-induced luminescence in lung, while ginger slightly decreased luminescent signals in
187
lung. Moreover, both ginger and zingerone slightly decreased LPS-induced luminescent
188
intensities in spleen.
189
We further analyzed the anti-inflammatory effects of ginger and zingerone in different
190
segments of small intestine. We divided the small intestine into 35 segments and the length
11
191
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
193
or zingerone was further represented as the inhibitory percentage. As shown in Figure 6, LPS
194
increased NF-κB-driven luminescence in whole small intestine, and the strong luminescence
195
was observed from segment 17 to 30, which corresponded to the region between mid-jejunum
196
and mid-ileum. Both ginger and zingerone suppressed LPS-induced bioluminescent intensity
197
in whole small intestine, and the inhibition from segment 14 to 35 was > 50% by ginger and
198
zingerone. Overall, these data suggested that ginger and zingerone displayed broad-spectrum
199
anti-inflammatory activities in most organs. Additionally, ex vivo imaging first showed that
200
LPS induced a more severe inflammation in the region spanning from mid-jejunum to
201
mid-ileum. Moreover, LPS-induced luminescent signal in the junction of jejunum and ileum
202
was suppressed efficiently by ginger and zingerone.
203
204
Ginger and Zingerone Inhibited LPS-Induced NF-κB Activation, IL-1β Production,
205
and Inflammatory Cell Infiltration in Small Intestine. IHC staining was further performed
206
to analyze the anti-inflammatory effects and mechanisms of ginger and zingerone in small
207
intestine. As shown in Figure 7, the number of IL-1β-positive cells was increased by LPS,
208
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,
12
210
including monocytes and granulocytes, while ginger and zingerone inhibited the expression
211
of CD11b-positive area. These data suggested that ginger and zingerone suppressed the
212
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
14
248
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
258
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.
16
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
300
301
SUPPORTING INFORMATION
302
HPLC profile of ginger extract (Supplementary Figure S1).
303
304
FUNDING SOURCES
17
immunosorbent
assay;
IHC,
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
310
311
NOTES
312
The authors declare no competing financial interest.
313
18
University
and
(CMU102-NSC-04
MOST
and
314
315
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FIGURE CAPTIONS
421
Figure 1. Ginger and zingerone. (A) Morphology of whole parts and cross sections of dried
422
ginger. (B) Chemical structure of zingerone.
423
424
Figure 2. Effects of ginger and zingerone on LPS-induced NF-κB activities in cells.
425
HepG2/NF-κB cells were treated with 100 ng/ml LPS and/or various amounts of ginger
426
extract and zingerone. MG-132 (5 µM) was used as a positive control. Twenty-four hours
427
later, NF-κB activity was measured by luciferase assay. Results are expressed as relative
428
NF-κB activity, which is presented as the comparison with RLU relative to solvent-treated
429
cells. Values are mean ± standard error (n=6). ###p < 0.001, compared with mock. **p <
430
0.01, ***p < 0.001, compared with LPS.
431
432
Figure 3. NF-κB-driven luminescence in living mice. (A) In vivo image. Transgenic mice
433
were administered with 1 mg/kg LPS and then treated with 100 mg/kg ginger or zingerone.
434
Four hours later, mice were injected intraperitoneally with D-luciferin and imaged for 1 min.
435
The color overlay on the image represents the photon/sec emitted from mice, as indicated by
436
the color scale. Photos are representative images (n=5/group). (B) Quantification of photon
437
emission from the whole body. Values are mean ± standard error. ###p < 0.001, compared
438
with mock. *p < 0.05, ***p < 0.001, compared with LPS.
25
439
440
Figure 4. Effects of ginger and zingerone on LPS-induced IL-1β and TNF-α production in
441
sera. Transgenic mice were challenged with 1 mg/kg LPS and then given with 100 mg/kg
442
ginger or zingerone. Four hours later, mice were sacrificed and the amount of IL-1β (A) and
443
TNF-α (B) in sera was quantified by ELISA. Values are mean ± standard error (n=5/group).
444
##p < 0.01, compared with mock. *p < 0.05, **p < 0.01, ***p < 0.001, compared with LPS.
445
446
Figure 5. NF-κB-dependent luminescence in individual organs. (A) Ex vivo imaging.
447
Transgenic mice were administered with 1 mg/kg LPS and then treated with 100 mg/kg
448
ginger or zingerone. Four hours later, mice were injected intraperitoneally with D-luciferin.
449
Five minutes later, mice were sacrificed, and organs were excised rapidly and subjected to
450
image (n=5/group). (B) Quantification of photon emission from organs. Values are mean ±
451
standard error. ##p < 0.01, ###p < 0.001, compared with mock. *p < 0.05, **p < 0.01, ***p <
452
0.001, compared with LPS.
453
454
Figure 6. Anti-inflammatory effects of ginger and zingerone in various segments of small
455
intestine. Transgenic mice were challenged with 1 mg/kg LPS and then given with 100 mg/kg
456
ginger or zingerone. Four hours later, mice were injected intraperitoneally with D-luciferin.
457
Five minutes later, mice were sacrificed, and organs were excised rapidly and subjected to
26
458
image. Small intestine was divided equally into 35 segments and the photon emission from
459
each segment was quantitated. Results are expressed as inhibition (%). Heatmap of small
460
intestine is shown on the bottom. The color overlay on the heatmap represents the photon/sec
461
emitted from each segment of small intestine, as indicated by the color scale.
462
463
Figure 7. IHC staining analysis of p65, IL-1β, and CD11b in jejunum. Transgenic mice were
464
challenged with 1 mg/kg LPS and then given with 100 mg/kg ginger or zingerone. Four hours
465
later, mice were sacrificed. Section of mid-jejunum was stained with antibodies against p65,
466
IL-1β, and CD11b (400× magnification). Photos are representative images (n=5/group)
27
Figure 1
(A)
(B)
28
Figure 2
5
Ginger
Relative NF-κB activity
###
Zingerone
**
4
*** ***
***
3
***
***
***
***
2
***
1
*** ***
***
***
0
Mock
LPS MG-132
0.5
1
2.5
5
10
Concentration (μg/ml)
29
50
100
Figure 3
(A)
Mock
LPS
LPS/Ginger LPS/Zingerone
photon/sec
(B)
###
400
Total flux (×106 photon/sec)
350
*
300
250
***
200
150
100
50
0
Mock
LPS
LPS/Ginger
30
LPS/Zingerone
Figure 4
(A)
800
##
IL-1β concetration (pg/ml)
700
600
500
400
300
**
***
200
100
0
Mock
LPS
LPS/Ginger
LPS/Zingerone
(B)
1,800
##
TNF-α concetration (pg/ml)
1,600
1,400
1,200
1,000
800
*
600
**
400
200
0
Mock
LPS
LPS/Ginger
31
LPS/Zingerone
Figure 5
(A)
Brain Heart
Lung
Liver Spleen Stomach Kidney
Intestine
Mock
LPS
LPS/Ginger
LPS/Zingerone
(B)
25000
###
Mock
LPS
20000
LPS/Ginger
Luminescent intensity
(×103 photon/sec/cm2/sr)
LPS/Zingerone
15000
*
###
###
10000
5000
**
###
*
***
*
**
**
*
###
##
*
###
###
**
0
Brain
Heart
Lung
Liver
32
Spleen
Stomach
Kidney
Intestine
0
33
ROI 35
ROI 34
ROI 33
ROI 32
ROI 31
ROI 30
ROI 29
ROI 28
ROI 27
ROI 26
ROI 25
ROI 24
ROI 23
ROI 22
ROI 21
ROI 20
ROI 19
ROI 18
ROI 17
ROI 16
ROI 15
ROI 14
ROI 13
ROI 12
ROI 11
ROI 10
ROI 9
ROI 8
ROI 7
ROI 6
ROI 5
60
ROI 4
ROI 3
ROI 2
ROI 1
Inhibition (o/o)
Figure 6
80
Ginger
Zingerone
40
20
Mock
LPS
LPS/Ginger
LPS/Zingerone
photon/sec
Figure 7
Mock
LPS
LPS/Ginger
p65
IL-1β
CD11b
34
LPS/Zingerone