Extract
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Introduction
Gastric carcinoma remains one of the most prevalent
malignant diseases in the world[1]. As there is no efficient
method for early diagnosis and therapy, the 5-year survival
rate for most patients with advanced gastric cancer is less
than 20%[2]. Atrophic gastritis is well known as a
premalignant disease with an increased risk in developing into
gastric carcinoma[3]. Therefore, several gastroenterologists have
emphasized the importance of intervention in atrophic
gastritis for the prevention of gastric
cancer[4].
Atrophic gastritis results from the long-term gastric
damages that impair the adaptive cytoprotection of gastric mucosa.
In recent years, the mechanism that protects the gastric
mucosa against intrinsic and extrinsic stimuli and maintains
the proper structure and function of the gastric mucosa has
attracted considerable attention[5]. Heat shock protein
(HSP)70, one of the major molecular chaperons, can adapt cells to
cope with various stresses in gastric
mucosa[6] and accelerate the cellular recovery from different stimuli by coping with
unfolded or denatured proteins[7]. The
overexpression of HSP70 has made cells resistant to death by increasing mucosal blood
flow in gastric mucosa[8,9].
Geranylgeranylacetone
(6,10,14,18-tetramethyl-5,9,13,17-nonadecatetraen-2-one, GGA; Eisai, Japan), an anti-ulcer drug,
protects gastric epithelial cells from
damage[10,11]. GGA has been shown to increase the protein level of HSP70 in cultured
guinea pig gastric mucosal cells and rat gastric mucosa while
preventing ethanol-induced gastric
damage[12]. In addition, the induction of HSP70 by GGA has found cytoprotective
action in a number of pathological lesions, including heart
ischemia[13],
hepatectomy[14], cerebral
infarction[15], and
colitis[16]. However, there is no literature regarding the
protective effect of GGA on atrophic gastritis. In this study, we
investigated whether GGA exerts its protective role in the
progression of atrophic gastritis via the induction of HSP70.
Materials and methods
Induction of atrophic gastritis in rats and treatment with
GGA Male Sprague-Dawley rats (200_300 g, Grade I) were
obtained from the Animal Center of Zhejiang Academy of
Medicine (Hangzhou, China). The study was in compliance
with the Declaration of Helsinki. The rats were maintained on a
12 h light/12 h dark cycle with food and water ad libitum.
Atrophic gastritis in rats was induced as reported
previously[17]. In brief, the rats were administered 0.1% ammonia solution, 60%
ethanol (1 mL/kg, ig on every Tuesday and Friday) and 20
mmol/L deoxycholic acid (1 mL/kg, ig every day) for 24 weeks.
Accompanied by the induction of atrophic gastritis, the rats
(n=6) were treated with GGA (200 mg/kg, ig) for 8 weeks, from
week 17 to the end of week 24. To further investigate whether
GGA protected gastric mucosa via the induction of HSP70,
we administered quercetin (100 mg/kg ig, n=6; Sigma, Saint
Louis, MO, USA), an inhibitor of HSP70
synthesis[18_20], in DMSO to rats daily for the final 8 weeks of the 24-week
period of atrophic gastritis induction. The control rats were
gavaged with DMSO (5 mL/kg, n=6). All the rats were then
sacrificed and the gastric tissues in lesser curvatures were
excised for further analyses. One half of each sample (within
1 cm from the pylorus) was immediately frozen in liquid
nitrogen and stored at -80 °C for the Western blot analysis. The
other half was fixed in 4% buffered formalin for histological
analysis and immunohistochemistry.
Inflammation index scoring in the antrum
The sections were stained with HE. A histological evaluation of the
severity of inflammation was performed by a scoring
criterion in accordance with the Sydney
system[21]. The inflammation score was assigned based on the following scale:
0=normal; 1=minimal inflammatory cells in pit or basal region
of gastric mucosa; 2=moderate number of inflammatory cells
mainly in two thirds of gastric mucosa; and 3=a large number
of inflammatory cells in gastric mucosa. Five random fields
of vision in the antrum were chosen in each section.
Quantitative analysis of gastric mucosal thickness and
gastric glands in the antrum Gastric mucosal thickness and
the quantity of gastric glands of 1 mm horizontal length were
measured with a micrometer eyepiece (Olympus Optical,
Tokyo, Japan) on HE-stained sections from the gastric
samples. Five random fields of vision in the antrum were
chosen in each section.
Immunohistochemistry evaluation The distribution of
HSP70 in gastric mucosa was assessed with
immuno-histo-chemistry. Tissues from rats were sectioned at 5
μm and mounted on poly L lysine coated slides. Antigen retrieval
was carried out by boiling sections in 10 mmol/L citrate buffer
(pH 6.0) for 10 min, and endogenous peroxidase activity was
blocked in methanol containing 3% hydrogen peroxide for
15 min. After blocking with 5% normal rabbit serum in
phosphate buffered solution (PBS) for 20 min, the sections were
incubated with mouse monoclonal antibody against human
HSP70 (1:1000, Sigma, USA) at 4 °C overnight. Non-specific
mouse immunoglobulin G (IgG) was applied as the negative
control. After washing in PBS, the sections reacted with the
biotinylated rabbit anti mouse IgG for 15 min at room
temperature and with the peroxidase-labeled streptavidin for
another 15 min. Then sections were stained with
diamino-benzidine-H2O2 solution for 3 min and counterstained with
hematoxylin.
Western blot analyses The total proteins isolated from
the gastric tissues were resolved by SDS-PAGE and
transferred to polyvinylidene difluoride (PVDF) membranes.
After blocking in 5% non-fat, dry milk, the membranes were
subjected with anti-HSP70 (1:6000, Sigma, USA) primary
antibody at 4 °C overnight and then washed 3 times. The
membranes were then incubated with horseradish
peroxidase-conjugated goat anti-mouse IgG (Santa Cruz
Biote-chnology, Santa Cruz, CA, USA) at a 1:5000 dilution for 1 h
at room temperature. The blotted membrane was visualized
by chemiluminescent substrate (EZ-ECL, Kibbutz Beit
Haemek, Israel). The immunoblotting for β-actin (1:1000,
Santa Cruz Biotechnology, USA) was used as a loading
control, and the densitometry was performed on Image-Pro
Plus 5.0 software (Media Cybernetics, Silver Spring, MD,
USA).
Statistical analysis Data were expressed as
median values. Differences between groups were examined for
statistical significance using non-parametric tests (median test).
P<0.05 denoted the presence of a statistical difference.
Results
Pathological changes in rats with atrophic gastritis
After 24 weeks of administration with ammonia solution,
ethanol, and deoxycholic acid, the rats developed atrophic
gastritis with an infiltration of lymphocytes into gastric
mucosa and loss of glandular cells in the gastric antrum (Figure
1). A quantitative analysis showed a significant increase of
the inflammation index in atrophic gastritis compared to that
in the controls (P<0.05; Table 1). The thickness of gastric
mucosa and the quantity of gastric glands were obviously
reduced in the rats with induced atrophic gastritis
(vs controls, P<0.05; Table 1).
Effect of GGA on the progression of atrophic gastritis in
rats Concomitant with the induction of atrophic gastritis in
rats, we administered GGA for 8 weeks and found that GGA
protected gastric mucosa from the continuous damages and
improved the pathological changes in gastric mucosa
compared with the atrophic gastritis group (Figure 1). GGA
decreased the inflammation index and significantly increased
the gastric mucosal thickness and glandular quantity (vs
atrophic gastritis, P<0.05; (Table 1). On the other hand,
quercetin accelerated the progression of atrophic gastritis in the
rats by significantly increasing the inflammatory cells in the
gastric antrum (vs vehicle control, P<0.05) and aggravating
the loss of glandular cells (Table 1).
Level of HSP70 expression in the gastric antrum of rats
Immunoblotting revealed a reduction of HSP70 expression
in the gastric antrum of rats with atrophic gastritis.
Treatment with 200 mg/kg GGA for 8 weeks significantly increased
the accumulation of HSP70, which was nearly 2-fold higher
than that in atrophic gastritis of rats
(P<0.05). In contrast, quercetin decreased the level of HSP70 in the gastric
mucosa compared with the vehicle-treated rats (Figure 2).
Distribution of HSP70 expression in the gastric antrum
HSP70 immunostaining was detectable in all gastric samples
from the rats by immunohistochemistry analyses.
Cytoplasmic staining was more frequent in the gastric epithelial cells
at the crest of mucosal folds. Conversely, nuclear staining
was mainly localized to the cells at the base of mucosal folds.
In the rats with atrophic gastritis, the nuclear
immunoreactivity of HSP70 was observed. The staining of HSP70 in the
GGA-treated rats was distributed in the whole gastric mucosa,
in both the nucleus and cytoplasm, but in the
quercetin-treated rats, HSP70 was mainly recognized in the cytoplasm
of the cells at crest of gastric mucosal folds, which was less
frequent than that in the vehicle control (Figure 3).
Discussion
Atrophic gastritis is characterized with chronic
inflammation of the gastric mucosa and loss of gastric glandular
cells with replacement by intestinal-type epithelium,
pyloric-type glands, and fibrous
tissue[22]. Damage to the gastric mucosa has been reported to correlate with the etiology of
atrophic gastritis. Helicobacter pylori (H pylori) infection is
by far the most common cause of chronic atrophic
gastritis[23]. The inoculation of H pylori in the gastric antrum caused
atrophic gastritis in the Japanese monkey model after 6
months of infection[24]. Epidemiology also revealed a
4.2-fold greater odds of atrophic gastritis for H pylori-positive
patients than H pylori-negative
patients[25]. H pylori produces ammonia in the stomach by the hydrolysis of urea,
which has an etiological role in H pylori-associated atrophic
gastritis[26]. In addition, bile reflux and alcohol consumption
could be potential risk factors for gastric atrophy and
intestinal metaplasia[25,27]. In this study, we integrated the
multiple factors including H pylori, bile, and ethanol to induce
atrophic gastritis in rats. As bile reflux diminished H pylori
from gastric mucosa[28], ammonia solution instead of live
H pylori was employed to simulate the conditions of
H pylori infection, and atrophic gastritis was successfully induced,
which showed the significant infiltration of inflammatory cells
and loss of glands in gastric mucosa. Moreover, our
previous study reported that the combined administration of 60%
ethanol, 20 mmol/L deoxycholic acid, and 0.5 g/L ammonia
for 12 weeks could induce gastritis with early features of
glandular atrophy in rats[17]. After 16 weeks of inducing
treatments, atrophic gastritis came into being in rats with
notable chronic inflammation and the loss of gastric
glandular cells. Further progression of atrophic gastritis was
observed after treatment for 24 weeks. Accordingly, GGA
was administered at week 17 when atrophic gastritis was just
induced and continued into week 24 when we investigated
the effect of GGA on the progression of atrophic gastritis.
In recent years, GGA has been reported to exert a
protective role in a variety of animal
models[12,16]. In mice with trinitrobenzene sulfonic acid-induced colitis, the
administration of GGA by oral gavage at 300 mg/kg suppressed
inflammation in the colons, and significantly improved mouse
survival rate[16]. GGA plays a cytoprotective role against acute
gastric mucosal lesions induced by
chemicals[29,30]. It has also been shown that GGA promotes the healing of acetic
acid-induced chronic gastric ulcers in
rats[31]. In this study, we demonstrated for the first time that the administration of
GGA in rats with atrophic gastritis results in protection
against further progression of atrophic gastritis. GGA
diminished the continuous damage to gastric mucosa and
facilitated histological recovery with inflammation relief and
glandular restoration.
One of the defense mechanisms of GGA has been
clarified to increase the expression of HSP70 to protect cells
against stresses. HSP70 is an important endogenous
cytopro-tective factor. GGA increased the protein level of
HSP70 in gastric mucosa of rats while preventing ethanol-induced
gastric damage[12]. The induction of HSP70 expression is
beneficial for preventing intestinal atrophy and maintaining mucosal
functions in intestinal cells[32]. Gastric atrophy results from
the long-term damage to gastric mucosa, and an aberrant
apoptosis is suggested to be involved in its
pathogenesis[33]. HSP70 is tightly related with the stability of cells to damage by
interference with apoptotic
programs[6,9]. In our study, the 2-fold level of HSP70 was induced by GGA in both the
cytoplasm and nucleus of gastric cells. HSP70 accumulation could
accelerate the recovery of atrophic gastritis via modulating
apoptosis and restoring the damaged cellular
structures[34,35]. Moreover, Pierzchaiski
has indicated that H pylori decreases the synthesis of HSP70 in gastric epithelial cells by the
inactivation of heat shock factor 1 in the recent
study[36]. The inhibition of HSP70 disturbed gastric adaptation and
facilitated H pylori to avoid host
immunity[37]. H pylori eradication, which has been reported to reverse the progression of
atrophic gastritis[38], is accompanied by increased HSP70
expression[39]. The induction of HSP70 by GGA might interrupt the
damage from H pylori. Additional studies are needed to
evaluate this relationship.
Quercetin is known to block the synthesis of HSP70 at
the level of mRNA accumulation[19] and then eliminates the
protective role of HSP70 to interrupt cell recovery from
damage[20,21]. Our data showed that quercetin suppressed the
accumulation of HSP70 in gastric cells, especially in the
nucleus, and increased the infiltration of inflammatory cells
in gastric mucosa. HSP70 depletion could aggravate
inflammation by significantly increasing the activation of
NF-κB and other inflammatory
cytokines[40]. The suppression of HSP70 was supposed to correlate with the progression of
atrophic gastritis, which further supported our findings that
HSP70 induced by GGA protected gastric mucosa from
continuous damage and facilitated the recovery of atrophic
gastritis in rats.
In conclusion, GGA prevents the progression of
atrophic gastritis via the induction of HSP70 expression.
Therefore, a potential drug target for the treatment of
atrophic gastritis is suggested.
Acknowledgement
We sincerely express our gratitude to Mr Yun-bin YAO,
Animal Laboratory of Medicine, Sir Run Run Shaw Hospital
(Hangzhou, China), for assisting with the animal model.
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