Materials and Methods
Animals
Male Wistar rats weighing 200-250 g were purchased from
Japan Clea (Tokyo, Japan) and housed in a regulated
environment. Standard laboratory chow and water were available
ad libitum. All experimental procedures were performed
according to the "Guidelines for the Care and Use of Laboratory
Animals" approved by each laboratory. Humane endpoints were
chosen to minimize or terminate pain or distress in the animals
via euthanasia, rather than waiting for death as an endpoint.
Tumor cell inoculation
The rat ascites hepatoma AH-130 cell line was kindly donated
by the Cell Resource Center for Biomedical Research, Institute of
Development, Aging and Cancer, Tohoku University, Japan. A
suspension of 108 AH-130 tumor cells was prepared in 2 ml
phosphate buffer saline and intraperitoneal (i.p.) injected into
rats.
Reagent preparation
Reagents of purity not less than 97% were used. Ghrelin (rat
acyl ghrelin, Peptide Institute, Osaka, Japan), (D-Lys3)-GHRP-6
(Bachem California, CA, USA), CRF (Peptide Institute) and
SB242084 dihydrochloride (Sigma, MO, USA) were dissolved in
saline or distilled water. Rikkunshito, a traditional herbal
medicine (spray-dried powder from herbal extracts, Tsumura,
Tokyo, Japan) was suspended in distilled water at doses of 125,
250, 500, and 1000 mg/kg for p.o. administration. Hesperidin
(LKT Laboratories, MN, USA) and atractylodin (Tsumura) were
dissolved in a 1% Tween-80 solution.
Sample preparation
Blood samples were collected using a tube containing
aprotinin and EDTA-2Na, and immediately transferred to a fresh
tube for a 3-min centrifugation at 10,000 rpm. For the assay of
acyl ghrelin, 10% 1mol/L HCl was added to the plasma obtained.
After processing, all sample aliquots were stored at -80C until
measurement. Total RNA was extracted from the hypothalamic
block using an RNeasy kit (Qiagen, CA, USA), and DNA was
removed from total RNA using RNase-Free DNase (Qiagen).
Reverse transcription reactions were performed using a TaqMan
reverse transcription kit (Applied Biosystems, CA, USA). All
oligonucleotide primers and fluorogenic probe sets for TaqMan
real-time PCR were obtained from Applide Biosystems; actin beta
(Rn00667869_m1), neuropeptide Y (Rn00561681_m1), agouti
related
peptide
(Rn01431703_g1),
proopiomelanocortin
(Rn00595020_m1),
cocaineand
amphetamine-regulated
transcript (Rn00567382_m1), orexin A (Rn00565995_m1),
melanin-concentrating hormone ([Pmch], Rn00561766_g1),
nesfatin-1 ([Nucb1], Rn01510621_m1), oxytocin (Rn00564446_g1),
urocortin-1 (Rn00569682_m1), urocortin-2,3 (Rn01445302_s1),
corticotropin-releasing factor (Rn01462137_m1), serotonin 2c
receptor (Rn00562748_m1).
Analytical assays
Levels of acyl ghrelin (Mitsubishi Chemical Medience, Tokyo,
Japan), C-reactive protein (CRP) (Life Diagnostics, PA, USA), and
CRF (Yanaihara Institute, Shizuoka, Japan) were measured
using an enzyme-linked immunosorbent assay. Leptin and
cytokine levels were measured using a rat serum adipokine
multiplex immunoassay (LINCOplex, LINCO Research, MO,
USA) and a rat cytokine 9-Plex assay (Bio-Plex, Bio-Rad, CA,
USA). Gene expression levels were measured using a real-time
polymerase chain reaction system (ABI 7900HT, Applied
Biosystems, CA, USA). β-smooth muscle actin mRNA was used as
an endogenous control.
Feeding tests
Rats were individually housed before feeding tests. Test drugs
were administered 5 days after tumor inoculation in rats.
Immediately, standard chow was provided and food intake was
measured over 2, 6, 24 or 48 hours. Concomitant administration
of (D-Lys3)-GHRP-6 (2 mol/kg) was performed immediately
before and 3 hours after the intake of chow.
Measurement of gastroduodenal motility
A strain gauge force transducer (F-08IS, Star Medical, Tokyo,
Japan) was placed onto the serosal surface of the rat antrum and
duodenum to measure circular muscle contractions.13
Measurements were made under free-moving conditions in
individual cages after a 5-day postoperative recovery period.
Electrophysiological study
Rats were anesthetized with urethane (1 g/kg, i.p.). A nerve
filament was dissected from the peripheral or central cut end of
the gastric branch of the vagus nerve to record afferent or efferent
nerve activity via a pair of silver wire electrodes. Dissection of
duodenal vagus nerve and adrenal sympathetic nerve filaments
was also performed to record these efferent nerve activities. A
rate meter with a rest time of 5 s was used to observe the time
course of nerve activity. Ghrelin at 1-1000 ng (0.3-300 pmol) was
administered through a small catheter inserted into the inferior
vena cava. Rikkunshito (1000 mg/kg) was administered through a
catheter inserted into the stomach or duodenum. The effects of
ghrelin and rikkunshito on the nerve activities were determined
by comparing the mean number of impulses per 5 s for 50 s before
and after the injection.
Measurement of ghrelin secretion in rats
Decapitation and blood sampling were performed 90 minutes
after i.p. administration of d-fenfluramine hydrochloride (5
mg/kg) to fasted rats. Oral administration of rikkunshito was
performed 120 minutes before blood sampling, and similar
procedures were used to measure the plasma concentrations of
acyl ghrelin.
Radioligand binding assays
A membrane protein fraction of Chinese Hamster Ovary
(CHO-K1) cells expressing human recombinant GHS-R was
prepared
in
25
mmol/L
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
buffer. The fraction was incubated with 0.03 nmol/L [125I]-ghrelin
containing rikkunshito (20–200 g/mL), its components (43
compounds, 10–100 mol/L), or vehicle (1% dimethylsulfoxide) for
60 min at 25°C. Nonspecific binding was estimated in the
presence of 100 nmol/L ghrelin and the amount of specifically
bound [125I]-ghrelin was determined.
Ca2+ imaging of GHS-1a receptor-expressing COS cells
transfected with the Ca2+ sensor G-CAMP2
Ca2+ imaging was performed using COS cells constitutively
expressing the GHS-1a receptor. Cells placed in 35 mm
glass-bottom dishes (1 x 104 cells/dish) were transfected with 0.05
g of G-CAMP2 cDNA. The fluorescence of G-CAMP2 was
continuously recorded at a wavelength of 510 nm, and the
fluorescence intensity in each whole cell was measured as
previously reported.54 Data are expressed as areas of fluorescence
intensity induced by rikkunshito compared with areas of those
induced by ghrelin alone, and were calculated as previously
reported.55
Measurements of [Ca2+]i in single neurons of the ARC or PVN
Single neurons isolated from the arcuate nucleus (ARC) or
paraventricular nucleus (PVN) of rats were superfused with 10
mmol/L
4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid
-buffered Krebs-Ringer bicarbonate buffers containing 10 and 1
mmol/L glucose for ARC and PVN, respectively. Cytosolic Ca2+
concentration ([Ca2+]i) was measured with fura-2 fluorescence
imaging as previously reported.19 Neurochemical identification of
neurons that exhibited [Ca2+]i responses was performed by
immunocytochemistry following the original method with slight
modifications.
Implantation of an i.c.v. catheter
Implantation of an i.c.v. catheter was performed 3 days before
AH-130 tumor inoculation. Anesthetized rats were placed in a
stereotaxic apparatus and the catheter was implanted with a
guide cannula (Eicom, Kyoto, Japan) that reached the right
lateral ventricle as described previously.15
Open-field test
Rikkunshito (500 mg/kg, p.o.) was administered twice daily
after tumor inoculation and the open-field test was performed on
day 5. Rats were placed in the central area of an open-field box
(90 cm width × 90 cm length × 30 cm height), and the behavior of
each rat was recorded on video for 600 s. The distance traveled by
each rat in 600 s was analyzed by a computer tracking program
(LimeLight, Actimetrics, IL, USA). The frequency of rearing
(standing on hind legs with or without contact with the sides of
the area) and grooming (using the paws or tongue to clean or
scratch the body) were also recorded. The fecal pellet output score
was assessed using a three-point scale: 3 = more than 4 pellets, 2
= 2 or 3 pellets, 1 = 1 pellet, 0 = no pellets; form of pellet: 1 = loose
and long (axis > 13 mm), 0 = normal.
Survival study
Test drugs were administered twice a day after AH-130 tumor
inoculation in rats. Cisplatin (CDDP; 1 mg/kg, i.p.) was
administered twice a week from 6 days.
Male BALB/c mice aged 7 weeks weighing 21-26 g were
purchased from Japan SLC (Hamamatsu, Japan). The CT-26
mouse colon carcinoma cell line (5 × 106 cells, ATCC, VA, USA)
was i.p. injected into mice. Mice were given rikkunshito (0.5 or
1.0%)-containing chow or control chow.
Human study
Patients who had pathologically proven stage III/IV pancreatic
cancer with ascites (n = 39, age; 66.4 ± 7.0 years, male/female;
23/16, plasma albumin; 3.8 ± 0.5 g/dL) were eligible candidates for
rikkunshito treatment, as suggested from clinical studies of this
drug in Japan. The patients were retrospectively analyzed from
2004 to 2009 in the Chiba Cancer Center. Rikkunshito (7.5 g, p.o.)
was given daily for more than 1 month and gemcitabine (1000
mg/m2) was administered in a 30-min intravenous infusion on
days 1, 8 and 15. The cycle was repeated every 28 days. Overall
survival was estimated from the date of the first treatment to
death.
Statistical analysis
Values for individual groups are shown as means ± standard
error (SE). To assess differences among groups, a Student t-test or
a multi-group Dunnett test was performed. Mortality data were
compared using Kaplan-Meier plots and Gehan-Breslow-Wilcoxon
tests. Values of P < 0.05 were considered statistically significant.
References
54. Lee MY, Song H, Nakai J, Ohkura M, Kotlikoff MI, Kinsey SP
et al. Local subplasma membrane Ca2+ signals detected by a
tethered Ca2+ sensor. Proc Natl Acad Sci USA 2006; 103:
13232–13237.
55. Gilbert D, Lecchi M, Arnaudeau S, Bertrand D, Demaurex N.
Local and global calcium signals associated with the opening of
neuronal alpha 7 nicotinic acetylcholine receptors. Cell
Calcium 2009; 45: 198–207.