Original Article

Yonago Acta medica 2016;59:100–110

Effect of Gingerol on Cisplatin-Induced Pica Analogous to Emesis Via Modulating
Expressions of Dopamine 2 Receptor, Dopamine Transporter and Tyrosine
Hydroxylase in the Vomiting Model of Rats
Weibin Qian,*† Xinrui Cai,‡ Yingying Wang,§ Xinying Zhang,† Hongmin Zhao,|| Qiuhai Qian,† Zhihong Yang,¶
Zhantao Liu¶ and Junichi Hasegawa**Division of Pharmacotherapeutics, Department of Pathophysiological and Therapeutic Science, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8503, Japan, †Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan
250011, Shandong, China, ‡Shandong Academy Occupational Health and Occupational Medicine, Shandong Academy of Medical Sciences, Jinan 250002, Shandong, China, §Jinan Shizhong People’s Hospital, Jinan 250002, Shandong, China, ||Hebei Province Cangzhou Hospital of Integrated Traditional and Western Medicine, Cangzhou 061000, Hebei, China and ¶Pharmic Department, Medical
College of Qingdao University, Qingdao 266021, Shandong, China

ABSTRACT

distribution of D2R, DAT and TH in the area postrema
and ileum were measured by immunohistochemistryand quantitated based on the image analysis, and the expression of DAT and TH in the area postrema and ileum
were measured by RT-PCR. The weights of kaolin eatenof the remaining rats were observed in every 6 h continuously for 72 h.
Results The weight of kaolin eaten in rats induced bycisplatin was significantly reduced by pretreatment withgingerol in a dose-dependent manner during the 0–24
h and 24–72 h periods (P < 0.05). Gingerol markedly
improved gastric emptying induced by cisplatin in adose-dependent manner (P < 0.05), and exhibited effective dose-dependent inhibition on the increase of expression levels of D2R and TH and the decrease of expression levels of DAT in both the ileum and area postrema (P< 0.05).
Conclusion Gingerol is effective on cisplatin-induced
emesis in rats possibly by inhibiting central or peripheral
increase of DA by inhibiting D2R, TH and accelerating
DAT.

Background

Gingerol, the generic term for pungent
constituents in ginger, has been used for treating vomiting in China. We are going to investigate the mechanisms of inhibitive effect of gingerol on cisplatin-induced pica behaviour by studying on both peripheral andcentral levels, and the effects of gingerol on homeostasisof dopamine (DA) transmission: dopamine D2 receptor(D2R), dopamine transporter (DAT) and tyrosine hydroxylase (TH).
Methods The antiemetic effect of gingerol was investigated on a vomiting model in rats induced by cisplatin
3 mg·kg−1 intraperitoneal injection (i.p.). Rats were randomly divided into the normal control group (C), simple
gingerol control group (CG), cisplatin control group(V), cisplatin + metoclopramide group (M), cisplatin +low-dose gingerol group (GL), cisplatin + middle-dose
gingerol group (GM) and cisplatin + high-dose gingerolgroup (GH). In observation period, rats in Groups C andV were pretreated with sterile saline 3 mL i.g.; rats inGroup CG were pretreated with gingerol 40 mg·kg−1 i.g.;
rats in Group M were pretreated with metoclopramide
2.5 mg·kg−1 i.g.; rats in Groups GL, GM and GH werepretreated with gingerol 10, 20 and 40 mg·kg−1 i.g. for 3days, respectively. Cisplatin (3 mg·kg−1, i.p.) was administered one time after each treatment with the antiemetic
agent or its vehicle except the Groups C and CG. The

Key words dopamine 2 receptor; dopamine transport;
gingerol; tyrosine hydroxylase; vomiting

It is generally accepted that nausea and vomiting (emesis) are components of protective mechanism by which
the human body defends itself against ingested toxins.1
However, chemotherapy-induced nausea and vomiting(CINV) is a distressing and common adverse event associated with cancer treatment2 that causes extreme discomfort and seriously impairs quality of patients’ life.3
Despite significant progress in the management of nausea and vomiting associated with chemotherapy, theseside effects continue to limit the effectiveness of cancer
therapy and weaken the quality of life of its victims.4
Cisplatin is one of the most effective and currently available chemotherapy agents. But it is also one of the mostCorresponding author: Qiuhai Qian, PhD
qianqiuhai@126.com
Received 2016 January 15
Accepted 2016 February 5
Abbreviations: 5-HT3, 5-hydroxytryptamine 3; C, normal control
group; CG, simple gingerol control group; CINV, chemotherapy-induced nausea and vomiting; D2R, dopamine D2 receptor;
DA, dopamine; DAT, dopamine transporter; GH, cisplatin + highdose gingerol group; GL, cisplatin + low-dose gingerol group;
GM, cisplatin + middle-dose gingerol group; i.g., intragastrically;i.p., intraperitoneal injection; M, cisplatin + metoclopramidegroup; SP, substance P; TH, tyrosine hydroxylase; V, cisplatin control group100

Gingerol ameliorates cisplatin-induced pica

is the generic term for pungent constituents in ginger.26 It
has been reported to inhibit contraction of isolated guinea-pig ileum by acting on 5-hydroxytryptamine 3 (5HT3) receptors and this is presumed to be related to its
antiememtic activity.27 During our previous research, wehave already proved that gingerol has effects on vomiting induced by cisplatin in minks, as well as prevented
the high expressions of DA.28, 29 But, the antiemetic
mechanism of gingerol remains unclear, and there are
few reports on the study of the homeostasis of DA transmission. Based on our previous studies above, we haveguessed that gingerol can adjust the abnormal expressionof DA in vomiting by inhibiting synthesis or accelerating
degradation of DA.
Therefore, in this study, we are going to investigate
the mechanisms of inhibitive effect of gingerol on cisplatin-induced pica behaviour by studying on both peripheral and central levels, and the effects of gingerol on
homeostasis of DA transmission: D2R, DAT and TH.

reasons on both acute and delayed emesis if anti-emetics are not given.5, 6 A lot of evidences are proved that
cisplatin can lead many adverse events such as CINV,
nephrotoxicity and peripheral neuropathy.7 Therefore,
the control of nausea and vomiting is critical for the patient’s quality of life and perception of chemotherapy.
Vomiting is caused by noxious stimulation directly
or indirectly on the vomiting center such as gastrointestinal tract, vestibular system, chemoreceptor trigger zone,
and higher centers in the cortex and thalamus. The nerve
impulse takes place when receptors are activated, andtransfer to the vomiting center, so emesis is initiated.8,
9The chemotherapy-induced emesis is developed via
peripheral and central systems, which are independent
relatively, and many neurotransmitters have been implicated in the pathogenesis of vomiting.10, 11 Biochemicalsignaling of emesis is much more diverse than previously believed.12 More recently, dopamine (DA) is believed
to be a major player in the motivational aspects of nausea and vomiting. The homeostasis of DA transmission
is mainly controlled by three regulatory elements: dopamine D2 receptor (D2R), DA transporter (DAT) andtyrosine hydroxylase (TH). D2R, one receptor of DA, is
known to activate the chemoreceptor trigger zone in thearea postrema of the fourth ventricle floor. This resultsin efferent impulses to the medullary vomiting centre,
which initiates vomiting.13, 14 DAT, a Na+/Cl− dependent
plasma membrane protein at nerve endings, can rapidly
reuptake DA, and determine the intensity and durationof DA transmission.15 TH, the rate-limiting enzyme forDA synthesis, participates in the regulation of intra- andextracellular DA levels.16, 17 However, studies of D2R,
DAT and TH were mainly concentrated in Parkinson’s
disease, obesity and so on.18–20 And researches of D2R,
DAT and TH on vomiting have not been involved.
Rats do not vomit. Instead, anticancer drugs induce
kaolin ingestion behavior “pica” in rats, and it is evaluated as an index of nausea/vomiting.21 Yamamoto et al.22
have reported that kaolin intake induced by anticancer
drugs in rats is related to their clinical emetogenic potential. In the present study, we investigated the effect ofgingerol on cisplatin-induced kaolin intake, which is anindex of nausea/vomiting in the rat.
Ginger, a classical traditional Chinese herbal medicine known as Zingiber officinale Rosce, has been used
for treating vomiting for 2000 years in China. It is acommon food for cooking for years in East Asia. Some
evidences have proved that ginger has effect on vomiting, post-operative nausea and vomiting and CINV.23–25
Studies with experimental animals (dogs and rats) haveshown that the various extracts of ginger and the ginger
juice possess anti-emetic effects against CINV. Gingerol

MATERIALS AND METHODS
Animals

Adult male Wistar rats weighing 200 ± 20 g were purchased from Shandong University Laboratory Animal
Shelter (Jinan, China). Rats were housed in individual
cages under controlled environmental conditions (22 ±2 °C relative humidity 40–60%, 12 h dark/light cycles,
food and water ad libitum). Animals were used in thepresent study in accordance with the guidelines of the
Shandong University Institutional Animal Care and Use
Committee. All studies were performed with approval
from the committee (2014012-KY).
Reagents

Gingerol (Xi’an Realin Biotechnology, Xi’an, China)
was dissolved in 1% tragacanth. Cisplatin (Qilu Pharmaceutical, Jinan, China) was prepared in normal saline
at 70 °C followed by gradual cooling to 40 °C and wasadministered immediately. Metoclopramide (Qilu Pharmaceutical, Jinan, China). Anti-D2R, anti-DAT, anti-TH,
and goat anti-rabbit antibody were obtained from Beijing
Bioss Biological Technology, Beijing, China.
Kaolin was prepared according to the method of theliterature,30 with minor modifications. Kaolin (Tianjin
Kemiou Chemical Reagent, Tianjin, China) was mixed
with 7% gum arabic (Tianjin Guangfu Fine Chemical
Research Institute, Tianjin, China) in distilled water toform a thick paste. The mixture was placed in a tube
and partially dried in a dryer. The mixture was extruded
from the tube, cut into a column of the same size as thatfor normal feed pellets, and dried completely in a dryer.
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W. Qian et al.

Animal experiments

Measurements of gastric emptying

105 rats were randomly divided into the following 7
groups: normal control group (C, n = 15), simple gingerol control group (CG, n = 15), cisplatin control group(V, n = 15), cisplatin + metoclopramide group (M, n =15), cisplatin+ low-dose gingerol group (GL, n = 15),
cisplatin+ middle-dose gingerol group (GM, n = 15) andcisplatin + high-dose gingerol group (GH, n = 15). In observation period, rats in Groups C and V were pretreatedwith sterile saline 3 mL i.g.; rats in Group CG werepretreated with gingerol 40 mg·kg−1 i.g.; rats in Group M
were pretreated with metoclopramide 2.5 mg·kg−1 i.g.;
rats in Groups GL, GM and GH were pretreated withgingerol 10, 20 and 40 mg·kg−1 i.g. for 3 days, respectively. Gingerol was dissolved with 1% tragacanth. In
the second night, rats have a 12 h fast. On the third day,cisplatin (3 mg·kg−1, i.p.) was administered 30 min after
each treatment with the antiemetic agent or its vehicle
except the Groups C and CG. After administration of
cisplatin, rats numbered 1 to 5 in each group were sacrificed to measure the gastric emptying 6 h later.
The weights of kaolin eaten of the remaining rats
were observed in every 6 h continuously for 72 h. To
measure the kaolin consumption during a 72 h period,
the remaining kaolin in the container and kaolin spilledin the cage were collected, dried, and weighed every 6 h.
Dosages of drugs were calculated based on bodysurface area. Adult patients take metoclopramide, gingerol, cisplatin 30 mg, 200 mg and 35 mg per day, respectively. The body surface area conversion coefficientfrom humans to rats is 0.018. Because drug metabolism
is not exactly the same between human and rat, we utilized 3 different doses of gingerol to evaluate its efficacy.
The concentrations of gingerols administered were calculated based on body surface area. Adult patients takegingerols at a dose of 200 mg per day, and the average
weight of an adult is 60 kg. The body surface area conversion coefficient from humans to rats is 0.018; thus thenormal dose (medium dose) of gingerols administered
to rats was 20 mg·kg−1. In order to evaluate the dose-depended effect, we use three different doses group. The
high dose is double the medium dose, and the low doseis half the medium dose.
The remaining rats were sacrificed 72 h after administration of cisplatin. Tissues from the area postrema
as well as the ileum were removed from rats. The rats
were placed in a stereotaxic frame (stoelting, Wood
Dale, IL) with their head positioned vertically according
to the Rat Brain Atlas by Paxinos and Watson.31 Stereotaxic coordinates of the area postrema: 0–3 mm lateral
to midline, 4.4–4.8 mm posterior to interaural line,
13.68–14.08 mm posterior to bregma.

Gastric emptying was adapted from the literatures withsome modifications.32, 33 Briefly, after a 12 h fast, rats
numbered 1 to 5 in each group were given 2 mL of thephenol red solution (0.5 mg/mL) mixed with 1.5% methylcellulose via gavage. Thirty minutes after the feeding, the rat was rapidly sacrificed under pentobarbital
anesthesia. The entire stomach was carefully isolated,
ligated just above the cardia and below the pylorus, andremoved. The amount of phenol red in the stomach was
determined by measuring the absorption at 560 nm using a spectrophotometer (Shenhua instrument and meterfactory, Shanghai, China). The percentage of gastric
emptying was defined as the ratio between the amountof phenol red recovered from the stomach and theamount of phenol red ingested into the stomach (1﹣theamount of phenol red recovered from the stomach/ theamount of phenol red ingested into the stomach).
Immunohistochemistry of D2R, DAT and TH

Tissues from the area postrema as well as the ileum
were removed from rats for immunohistochemical analysis of D2R, DAT and TH. The ileum was excised at a
distance of 20 cm from the pylorus. The samples were
fixed in 10% buffered formalin at 4 °C for 24–48 h.
This was followed by cryoprotection for 12–18 h at 4 °C
using 30% sucrose or until the samples equilibrated. The
samples were sectioned (4 μm thick) using a freezing
microtome (ASpZr35) and standard immunohistochemical procedures were used to visualize D2R, DAT and
TH protein. Sections were washed 4 × 10 min in PBS (pH7.4) at room temperature and were blocked with 10%
normal goat serum. Sections were then incubated with
anti-D2R, anti-TH or anti-DAT (Beijing Bioss Biological Technology, Beijing, China) overnight on a shaker
at room temperature. Following washing (4 × 10 min)with PBS, sections were incubated for 2 h with goat anti-rabbit antibody. Finally, sections were washed (3 × 10min) in PBS, transferred onto gelatin-coated slides. Tissue processed without the primary D2R antibody, DAT
antibody of TH antibody served as a negative control.
Adjacent sections were mounted onto plus-coated slides
and stained using Neutral Red (0.5%), washed through aseries of increasing alcohol concentrations, cleared withxylene and coverslipped with Neutral Blasam.
Five fields from every section were randomly selected for examination using an Olympus CKX41-32PH
microscope equipped with an imaging system (Olympus
Optical, Japan). The distribution and staining of positive
expression of the area postrema and ileum tissue wereobserved under 200 light microscopes. The optical densities were measured by Image-Pro Plus v 6.0 software(Media Cybernetics, Rockville, MD).
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Gingerol ameliorates cisplatin-induced pica

RT-PCR of DAT and TH

second step, quantitative real-time PCR was performedon the LightCycler apparatus (Roche Diag Diagnostics,
Mannheim, Germany) using Power SYBR Green. PCR
Master Mix kits (Applied Biosystems). The reaction
was conducted with an initial denaturing at 95 °C for
10s, and then involved 40 cycles of 55 °C 10 s, 72 °C
15 s, and terminated by a cooling step 30 s at 40 °C. A
melting-curve analysis was performed to confirm theabsence of primer dimmers in specific PCR products.
The efficiency of RT-PCR was assessed with serial dilutions of a sample of cDNA from the normal control
group. Each experiment was performed in duplicate andthe data were analyzed using LightCycler Software 4.0(Roche Diagnostics). Beta-actin mRNA was used as thehousekeeping gene, and all data are represented relative
to its expression as fold change from control group.

To measure mRNA expressions of DAT and TH in
the area postrema and ileum tissue, the tissues in rats
were quickly immersed in RNAlater and stored at –80°C. The total RNA was sequentially extracted using
TRIZOL Reagent (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instruction. The RNA
was treated with DNase (DNAfree, Ambion, Austin,TX) in order to remove contaminating genomic DNA,followed by phenol, chloroform extraction and ethanol
precipitation. Total RNA was assessed for purity using
the NanoDrop system. Total RNA was performed in a
two-step procedure as described by Power SYBR GreenPCR Master Mix kits (Applied Biosystems). Briefly, inthe first step, cDNA was prepared from 500 ng RNA
by reverse transcription in a final volume of 20 μL in athermal cycler (Tgradient 96, Whatman Biometra, Niedersachsen, Germany). The samples were incubated at
37 °C for 60 min and 95 °C for 5 min. The cDNA wasstored at –20 °C. The rat-specific primers for the genes
of PPT, NEP and beta-actin were designed using Primer Premier 5.0 (Premier Biosoft, Palo Alto, CA). The
primer sequences are shown in Table 1. Primers weresynthesized by BioAsia Corp. (Shanghai, China). In theStatistical analysis

All statistical analyses were performed using SPSS 17.0
software (SPSS, Chicago, IL). Data were expressed asmean ± S.E. Analysis of variance (ANOVA) was performed when more than two groups were compared.
Values of P < 0.05 were considered statistically significant.

Table 1. The sequence of each PCR primer
Gene

Forward

ReverseBeta-actin

5’- GAGGGAAATCGTGCGTGAC -3’

5’- GGACTCATCGTACTCCTGCTTG -3’

TH

5’- GTGTTTCAATGCACCCAGTAT -3’

5’- GTCCAATGTCCTGGGAGA -3’

DAT

5’- CTGCTCTACTTCAGCCTAT -3’

5’- GCATGGTAGCTGTGATCC -3’

DAT, dopamine transporter; PCR, polymerase chain reaction; TH, tyrosine hydroxylase.

RESULTS

Gastric emptying

Pica induced by cisplatin

Gastric emptying was significantly delayed after administration with cisplatin. As shown in Fig. 2, the percentage of gastric emptying was significantly decreased inGroup V compared with Groups C and CG (P < 0.05),
and the delayed gastric emptying was significantly improved after the treatment with metoclopramide andgingerol in comparison with Group V (P < 0.05). Metoclopramide had better effect than low-dose and middle-dose gingerol (P < 0.05). Gingerol improved gastric
empting in a dose dependent manner (P < 0.05).

As shown in Fig. 1, rats dosed with vehicle followedby cisplatin had two distinct periods of pica behaviour.
There was an initial acute phase in 24 h after administration with cisplatin. After this period there was a delayed phase last near 48 h in Group V. Metoclopramide
significantly inhibited the pica response during 24 hfollowing cisplatin injection (P < 0.05). However, there
was no significant decrease of kaolin eaten during 24–72
h. Gingerol dose-dependently decreased the weight ofkaolin eaten induced by cisplatin during the 72 h observation period with significant decreases on each of
the three test days (P < 0.05). And simple gingerol did
not significantly induce the pica behavior, there was no
significant difference in the weight of kaolin eaten withGroup C.

D2R, TH and DAT immunostaining expression inarea postrema and ileum

D2R (Fig. 3A), TH (Fig. 4A) and DAT (Fig. 5A) staining
intensities (Red-brown deposits indicate positive staining) were mainly situated in the mucosa and submucosa
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Kaolin eaten (mg)of the ileum as well as in the neurons of the area postrema.
Compared with Group C and CG, there were more D2R,
TH and less DAT immunoexpressions detected in GroupV. Metoclopramide and gingerol had significant effecton the expression of D2R, TH and DAT in both the area
postrema and ileum. Gingerol markedly suppressed
the amount of D2R and TH detected and increased theamount of DAT detedted in a dose-dependent manner (×200).
Mean optical density values of D2R, TH and DAT
are showed in Figs. 3B, 4B and 5B, respectively. The
photographs generated were quantitatively analyzed theoptical density of D2R, TH and DAT by Image-Pro Plus.
D2R and TH expressions significantly increased after
treated with cisplatin while DAT expressions decreased(P < 0.05). Metoclopramide and gingerol had significant
reducing effect on the elevated expressions of D2R, TH
and elevation effect on the increased expressions of DAT
in both the area postrema and ileum (P < 0.05). The
increase of D2R, TH expressions and decrease of DAT
expression in the area postrema and ileum induced bycisplatin was significantly inhibited by gingerol in a dose
dependent manner (P < 0.05). Metoclopramide had better effect than low-dose and middle-dose gingerol on theexpression of D2R (P < 0.05); had better effect than lowdose gingerol and weaker effect than high-dose gingerol
on the expression of TH (P < 0.05); had weaker effectthan high-dose gingerol on the expression of DAT (P <0.05) both in the area postrema and ileum.

Time (h)Fig. 1. Pica induced by cisplatin.
Rats had two distinct periods of eating kaolin in the cisplatingroups, and Ginger significantly decreased the weight of kaolin
eaten induced by cisplatin during the 72 h observation period.
Metoclopramide significantly decreased the weight of kaolin eaten
during 24 h following cisplatin injection, but there was no significant decrease in the pica during 24–72 h. C: normal control group,
pretreated with sterile saline 3 mL every day (n = 10); CG: simple
gingerol control group, pretreated with gingerol 40 mg·kg−1 i.g.
every day (n = 10); V: cisplatin control group, pretreated with sterile saline 3 mL every day (n = 10); M: cisplatin + metoclopramidegroup, pretreated with metoclopramide 2.5 mg·kg−1 i.g. everyday (n = 10); GL: cisplatin + low-dose gingerol group, pretreatedwith gingerol 10 mg·kg−1 i.g. every day (n = 10); GM: cisplatin +middle-dose gingerol group, pretreated with gingerol 20 mg·kg−1
i.g. every day (n = 10); GH: cisplatin + high-dose gingerol group,
pretreated with gingerol 40 mg·kg−1 i.g. every day (n = 10). i.g.,
intragastrically.

mRNA expression of TH and DAT in area postrema
and ileum

As shown in Fig. 6, TH mRNA expression in both area
postrema and ileum in Group V was significantly increased while DAT decreased compared to that in othergroups (P < 0.05). Metoclopramide and gingerol could
decrease TH mRNA expression (P < 0.05) and increase
DAT mRNA expression (P < 0.05). Metoclopramide
had better effect than low-dose gingerol (P < 0.05). Gingerol decrease TH mRNA expression and increase DATmRNA expression in a dose dependent manner in botharea postrema and ileum (P < 0.05).

Fig. 2. Gastric emptying.
C: normal control group, pretreated with sterile saline 3 mL everyday (n = 5); CG: simple gingerol control group, pretreated withgingerol 40 mg·kg−1 i.g every day (n = 5); V: cisplatin control
group, pretreated with sterile saline 3 mL every day (n = 5); M:
cisplatin + metoclopramide group, pretreated with metoclopramide 2.5 mg·kg−1 i.g. every day (n = 5); GL: cisplatin + low-dose
gingerol group, pretreated with gingerol 10 mg·kg−1 i.g. every day (n= 5); GM: cisplatin + middle-dose gingerol group, pretreated withgingerol 20 mg·kg−1 i.g. every day (n = 5); GH: cisplatin + highdose gingerol group, pretreated with gingerol 40 mg·kg−1 i.g. everyday (n = 5). *P < 0.05 vs. Group C, ‡P < 0.05 vs. Group CG, †P <0.05 vs. Group V, §P < 0.05 vs. Group M, ||P < 0.05 vs. Group GL,¶P < 0.05 vs. Group GM. i.g., intragastrically.

DISCUSSION
Nausea and vomiting are complex protective mechanisms. However, when these symptoms recur frequently,
they can significantly reduce the quality of life and canalso be detrimental to health.34 Chemotherapy can be alife-prolonging treatment for many cancer patients, but
it is often associated with profound nausea and vomitingthat is so distressing.35 Despite advances in anti-emetic
therapy, chemotherapy-induced nausea and vomiting
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Gingerol ameliorates cisplatin-induced pica

still poses a significant burden to patients undergoing
chemotherapy.36 Metoclopramide, as a clinical drug of
D2R antagonists, has effects on vomiting and gastrointestinal diseases.37 It is reported that metoclopramide,
given in doses of 10 mg, has been shown to an effective
prophlaxis against vomiting.38 However, a recent meta-analysis showed no statistically significant amountof extrapyramidal symptoms, dizziness, headache, orsedation with this dose of metaclopramide1.39 Traditional Chinese medicines have attracted attention due
to their distinctive biological activities without toxicity
and/or side-effects.40 Gingerol has been shown to beeffective antiemetics on both acute and delayed emesis
in our previous studies.41, 42 In this study, the amount ofconsumption of kaolin was significantly reduced afteradministration with gingerol compared with the control group during the whole 72 h. And gingerol has adose-dependent effect. Therefore, we had investigated
the antiemetic efficacy of gingerol on cisplatin-induced
vomiting model of rats. In this study, gastric emptying
was significantly delayed after administration with cisplatin and the delayed gastric emptying was significantly
improved after the treatment with metoclopramide andgingerol. Gingerol improved gastric empting in a dose
dependent manner.
Dopamine receptors, specifically D2 and D3, are
known to play a role in nausea and emesis, most likely
through inhibition of adenylate cyclase,43 which alters
the amount of cAMP within neurons located in thearea postrema.44 D2R, one receptor of DA, is known

Fig 3.
A
A

B
Optical density

C
CG
V
M
GL
GM
GH
Area Postrema

Ileum

Fig. 3. D2R immunostaining expression in area postrema and ileum.
A: Immunohistochemistry expression of D2R in area postrema and ileum. The figures above (C1, CG1, V1, M1, GL1, GM1, GH1) show
the expression in area postrema. The figures below (C2, CG2, V2, M2, GL2, GM2, GH2) show the expression in ileum. B: Mean opticaldensity values of D2R. The photographs generated were quantitatively analyzed the optical density of D2R with Image-Pro Plus. C: normal control group, pretreated with sterile saline 3 mL every day (n = 10); CG: simple gingerol control group, pretreated with gingerol 40
mg·kg−1 i.g. every day (n = 10); V: cisplatin control group, pretreated with sterile saline 3 mL every day (n = 10); M: cisplatin + metoclopramide group, pretreated with metoclopramide 2.5 mg·kg−1 i.g. every day (n = 10); GL: cisplatin + low-dose gingerol group, pretreated with−1
gingerol 10 mg·kg i.g. every day (n = 10); GM: cisplatin + middle-dose gingerol group, pretreated with gingerol 20 mg·kg−1 i.g. every day (n= 10); GH: cisplatin + high-dose gingerol group, pretreated with gingerol 40 mg·kg−1 i.g. every day (n = 10). *P < 0.05 vs. Group C, ‡P <0.05 vs. Group CG, †P < 0.05 vs. Group V, §P < 0.05 vs. Group M, ||P < 0.05 vs. Group GL, ¶P < 0.05 vs. Group GM. D2R, dopamine
D2 receptor; i.g., intragastrically.

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W. Qian et al.

to activate the chemoreceptor trigger zone in the area
postrema of the fourth ventricle floor. This results inefferent impulses to the medullary vomiting centre,
which initiates vomiting.13, 14 Metoclopramide is a potent
D2Receptor antagonist and binds to D2 receptors in thearea postrema.43 We have already proved that gingerol
can reduce the expression of DA in minks.29 In thisstudy, the expressions of D2R was increased after injectwith cisplatin in both the mucosa and submucosa of theileum as well as in the neurons of the area postrema.
Metoclopramide and gingerol had significant effect on
the expression of D2R in both the area postrema and ileum. Gingerol markedly suppressed the amount of D2R
detected in a dose-dependent manner. We have got theconsistent conclusion with our previous study that gin-

gerol can significantly inhibit cisplatin-induced vomiting
by improve the increased D2R expression on both area
postrema and ileum.
DAT, a Na+/Cl− dependent plasma membrane protein at nerve endings, can rapidly reuptake DA, and determine the intensity and duration of DA transmission.15
It is extensively distributed in peripheral and central nervous system and participated in many physiological processes. K Lemmer has proved that DAT can up-regulate
the expression of DA in neuroendocrine gastrointestinal
tumor cells.45 The expression of DAT is associated withsome clinical diseases, such as Parkinson’s disease,46
and is known to play an important role in gastrointestinal physiology.47 But it has not been investigated in
CINV so far. In this study, we have measured expression

Fig 4.
A
A

B
Optical density

C
CG
V
M
GL
GM
GH
Area Postrema

Ileum

Fig. 4. TH immunostaining expression in area postrema and ileum.
A: Immunohistochemistry expression of TH in area postrema and ileum. The figures above (C3, CG3, V3, M3, GL3, GM3, GH3) show
the expression in area postrema. The figures below (C4, CG4, V4, M4, GL4, GM4, GH4) show the expression in ileum. B: Mean opticaldensity values of TH. The photographs generated were quantitatively analyzed the optical density of TH with Image-Pro Plus. C: normal control group, pretreated with sterile saline 3 mL every day (n = 10); CG: simple gingerol control group, pretreated with gingerol 40
mg·kg−1 i.g. every day (n = 10); V: cisplatin control group, pretreated with sterile saline 3 mL every day (n = 10); M: cisplatin + metoclopramide group, pretreated with metoclopramide 2.5 mg·kg−1 i.g. every day (n = 10); GL: cisplatin + low-dose gingerol group, pretreated withgingerol 10 mg·kg−1 i.g. every day (n = 10); GM: cisplatin + middle-dose gingerol group, pretreated with gingerol 20 mg·kg−1 i.g. every day (n= 10); GH: cisplatin + high-dose gingerol group, pretreated with gingerol 40 mg·kg−1 i.g. every day (n = 10). *P < 0.05 vs. Group C, ‡P < 0.05
vs. Group CG, †P < 0.05 vs. Group V, §P < 0.05 vs. Group M, ||P < 0.05 vs. Group GL, ¶P < 0.05 vs. Group GM. i.g., intragastrically; TH,tyrosine hydroxylase.

106Gingerol ameliorates cisplatin-induced pica

correlation on Parkinson disease48 and gastroparesis.49
In the present research, we measure the expression of
TH by both immunohistochemistry and RT-PCR. The
results illustrate that metoclopramide and gingerol could
decrease TH immunostaining expression and mRNA
expression. By considering the similar changes of D2R
and TH, we have inferred that gingerol has effect on
D2R, TH and DAT to regulate the abnormal expressionof DA on the treatment of vomiting induced by cisplatin
in rats.
In present findings, gingerol has showed efficacy
effects on vomiting induced by cisplatin. Gingerol could
efficiently reduce DA expression by inhibiting synthesis
and accelerating decomposition by adjusting mRNA
associated with DA, such as D2R, DAT and TH. On

of DAT to evaluate the mechanism why gingerol can improve vomiting by regulating the abnormal expression of
DA. DAT mRNA expression in both area postrema andileum in Group V was significantly decreased comparedto that in other groups. Metoclopramide and gingerol
could increase it. Gingerol increase DAT mRNA expression in a dose dependent manner in both area postrema
and ileum. And the expression of DAT has positive correlation with DA.
Except D2R and DAT, we have also analyzed TH
to explore the mechanism of DA and vomiting. A lot of
enzymes are involved in the metabolism of the DA. TH,
the rate-limiting enzyme for DA synthesis, participatesin the regulation of intra- and extracellular DA levels.16,17Some evidences reveal that TH and DA have negative

Fig 5.
A
A

B
Optical density

C
CG
V
M
GL
GM
GH
Area Postrema

Ileum

Fig. 5. DAT immunostaining expression in area postrema and ileum.
A: Immunohistochemistry expression of DAT in area postrema and ileum. The figures above (C5, CG5, V5, M5, GL5, GM5, GH5) show
the expression in area postrema. The figures below (C6, CG6, V6, M6, GL6, GM6, GH6) show the expression in ileum. B: Mean opticaldensity values of DAT. The photographs generated were quantitatively analyzed the optical density of DAT with Image-Pro Plus. C: normal control group, pretreated with sterile saline 3 mL every day (n = 10); CG: simple gingerol control group, pretreated with gingerol 40
mg·kg−1 i.g. every day (n = 10); V: cisplatin control group, pretreated with sterile saline 3 mL every day (n = 10); M: cisplatin + metoclopramide group, pretreated with metoclopramide 2.5 mg·kg−1 i.g. every day (n = 10); GL: cisplatin + low-dose gingerol group, pretreated withgingerol 10 mg·kg−1 i.g. every day (n = 10); GM: cisplatin + middle-dose gingerol group, pretreated with gingerol 20 mg·kg−1 i.g. every day (n= 10); GH: cisplatin + high-dose gingerol group, pretreated with gingerol 40 mg·kg−1 i.g. every day (n = 10). *P < 0.05 vs. Group C, ‡P <0.05 vs. Group CG, †P < 0.05 vs. Group V, §P < 0.05 vs. Group M, ||P < 0.05 vs. Group GL, ¶P < 0.05 vs. Group GM. DAT, dopaminetransporter; i.g., intragastrically.

107

TH against beta-action

DTA against beta-action

W. Qian et al.

Area Postrema

Ileum

C
CG
V
M
GL
GM
GH
Area Postrema

Ileum

Fig. 6. Changes of mRNA expression of TH and DAT in area postrema and ileum.
Levels of TH and DAT against beta-actin mRNA expression are shown in the above histogram. The left-side graphs show the expressionin area postrema. The right-side graphs show the expression in ileum. C: normal control group, pretreated with sterile saline 3 mL everyday (n = 10); CG: simple gingerol control group, pretreated with gingerol 40 mg·kg−1 i.g. every day (n = 10); V: cisplatin control group,
pretreated with sterile saline 3 mL every day (n = 10); M: cisplatin + metoclopramide group, pretreated with metoclopramide 2.5 mg·kg−1−1i.g. every day (n = 10); GL: cisplatin + low-dose gingerol group, pretreated with gingerol 10 mg·kg i.g. every day (n = 10); GM: cisplatin−1+ middle-dose gingerol group, pretreated with gingerol 20 mg·kg i.g. every day (n = 10); GH: cisplatin + high-dose gingerol group, pretreated with gingerol 40 mg·kg−1 i.g. every day (n = 10). *P < 0.05 vs. Group C, ‡P < 0.05 vs. Group CG, †P < 0.05 vs. Group V, §P < 0.05
vs. Group M, ||P < 0.05 vs. Group GL, ¶P < 0.05 vs. Group GM. DAT, dopamine transporter; i.g., intragastrically; TH, tyrosine hydroxylase.

REFERENCES

the other hand, the effect of gingerol showed stronger
effects than metaclopramide on expressions of DAT and
TH when using at the higher dosage. According to thatmetaclopramide is a clinical drug of D2R antagonists
on emesis,37 as well as amount of side effects,39 we
speculate that gingerol has more comprehensive effects
than metaclopramide on emesis and vomiting, while
gingerol can regulate the abnormal expression of DA byinhibiting synthesis and accelerating decomposition byadjusting mRNA or enzymes associated with DA, suchas D2R, DAT and TH.
Nevertheless, the reason why gingerol can modulate
expression of D2R, DAT and TH not only in the ileum
but also area postrema remains unclear. Because themRNA levels were changed, it might be modulated in
the transcriptional step or others. This research might be
partly proved the effect on cisplatin-induced pica analogous to emesis via modulating expressions of D2R, DAT
and TH in the vomiting model of rats. For the further
more study of underlying molecular biological mechanisms, we will use the Western blot method combinewith RT-PCR method in the next experiment.

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