Effect of Areca nut on Helicobacter pylori-induced gastritis in mice · 2020-07-03 · Effect of...
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Effect of Areca nut on
Helicobacter pylori-induced gastritis in mice
Jinwook Lee
The Graduate School
Yonsei University
Department of Dentistry
Effect of Areca nut on
Helicobacter pylori-induced gastritis in mice
Directed by Professor In-Ho Cha
A Dissertation Submitted to
the Department of Dentistry
and the Graduate School of Yonsei University
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
Jinwook Lee
June 2016
This certifies that the Dissertation
of Jinwook Lee is approved.
___________________________
Thesis Supervisor: In-Ho Cha
___________________________
Yun-Jung Yoo
___________________________
Hyung-Jun Kim
___________________________
Jeong-Heon Cha
___________________________
Woong Nam
The Graduate School
Yonsei University
June 2016
i
CONTENTS
LIST OF FIGURES ·········································· iii
ABSTRACT ·················································· iv
I. INTRODUCTION ········································· 1
II. MATERIALS AND METHODS ······················· 7
1. Diet and water preparation ·················································· 7
2. Animal experiments ·························································· 7
3. H. pylori culture and Mouse infection ····································· 11
4. Determination of the number of viable H. pylori in stomach ··········· 11
5. Histological analysis ························································· 12
6. Statistical analysis ····························································· 13
ii
III. RESULTS ·················································· 14
1. Establishment of mouse model for H. pylori-induced gastritis
development ···································································· 14
2. Gross changes of food intake and body weight of mice ·················· 19
3. Effect of AN and MNU on viable H. pylori colonization in the H.
pylori-infected mouse stomach ·············································· 21
4. Effect of AN and MNU on the stomach weight of H. pylori-infected
mice.············································································· 21
5. Effect of AN and MNU on gastric lesions of H. pylori-infected mice ··· 25
IV. DISCUSSION ············································· 30
V. REFERENCES ············································· 34
ABSTRACT (IN KOREAN) ································ 42
iii
LIST OF FIGURES
Figure 1. Experimental design ················································· 9
Figure 2
.
Colonization of H. pylori PMSS1 in mice stomach after 4
weeks and 12 weeks from infection ······························· 15
Figure 3. Histopathological evaluation of mice stomach after H. pylori
infection ······························································· 16
Figure 4. Gross change of food intake and body weight in mice during
the experimental period ············································· 20
Figure 5. The viable number of H. pylori colonization in the gastric
mucosa and the ratio of stomach weight to body weight in H.
pylori infected mice ·················································· 22
Figure 6. Effect of Areca nut and MNU on H. pylori induced gastric
lesions of mice ························································ 26
iv
ABSTRACT
Effect of Areca nut on Helicobacter pylori-
induced gastritis in mice
Jinwook Lee
Department of Dentistry
The Graduate School, Yonsei University
(Directed by Professor In-Ho Cha)
Areca nut (AN) chewing is a habit of humans in many countries in the Central,
Southern and South-east Asia. It is strongly associated with the occurrence of oral,
pharyngeal, and esophageal cancer as well as systemic inflammation. However,
the association between AN intake and development of gastric lesion has not been
identified yet. The aim of this study is to investigate the effect of AN on gastric
diseases using a mouse model for H. pylori infection. There were seven groups;
mice fed with normal diet (ND), mice fed with 2.5% AN-containing diet (AD),
mice fed with 200 ppm MNU-containing water (MNU), mice fed with normal diet
and infected with H. pylori PMSS1 strain (ND/HP), mice fed with 2.5% AN-
containing diet and infected with H. pylori PMSS1 strain (AD/HP), mice fed with
200 ppm MNU-containing water and infected with H. pylori PMSS1 strain
(MNU/HP), and mice fed with 2.5% AN-containing diet, 200 ppm MNU-
containing water and infected with H. pylori PMSS1 strain (AD/MNU/HP). Food
v
intake and body weight gain were monitored weekly during experiments and mice
were sacrificed at 10-week after the infection. After sacrifice, stomach weight, H.
pylori colonization, and gastric inflammation were determined. The stomach
weight was significantly increased in H. pylori-infected groups with the increase
in H. pylori colonization, but the two groups showed no significant difference of
the stomach weight and the colonization. In histological grading, mononuclear
cells infiltration in AD/HP group was more severe than that in ND/HP group.
These data suggest that chronic gastric inflammation was aggravated by AN
treatment in H. pylori-induced gastric lesion. Furthermore, this animal model will
be useful for future experiments to determine the effect of potential carcinogens
on H. pylori-induced gastric lesions.
Key words: Areca nut, Helicobacter pylori, Gastritis, Oral cancer
1
Effect of Areca nut on Helicobacter pylori-
induced gastritis in mice
Jinwook Lee
Department of Dentistry
The Graduate School, Yonsei University
(Directed by Professor In-Ho Cha)
I. INTRODUCTION
Areca nut (AN) is the fruit seed of the palm tree, Areca catechu. It is often mixed
with betel leaf with or without tobacco to make betel quid, which is used as a
masticatory substance by approximately 600 million people worldwide (Gupta
and Warnakulasuriya, 2002). Tobacco chewing combining AN, betel leaf and
slaked lime together with tobacco is also a common tobacco-related habit in many
Asian countries including the Central, Southern and South-east Asia (IARC, 2004).
Usage of AN along with betel leaves is a cultural and traditional symbol in Indian
2
subcontinent where as its chewing dates back to the pre-Vedic period of Harappan
Empire (Prabhu et al., 2014).
Fresh AN husk is a green color fruit containing soft nuts. With ripening, the husk
becomes yellow or orange while hardening its inside. The seed of AN can be
consumed fresh, boiled or after drying in the sun (Angadi and Rao, 2011). The
constituents of the AN are carbohydrate, fat, protein, crude fibre, polyphenol such
as flavonol and tannin, major alkaloid arecoline, and mineral matters (Kumar et
al., 2015). It is consumed by people of all the age groups and AN chewing is a
popular habit in Asian and Oceanic countries. It is considered to be commonly
used a psychoactive substance such as tobacco, alcohol, and caffeine (Nelson and
Heischober, 1999).
The chewing of betel quid brings a mild stimulant effect to the central nervous
system (Gupta and Ray, 2004). Also it increases salivation which is useful for
digestion. AN has a medicinal property of deworming and preventing halitosis
together with betel leaf (Prabhu et al., 2014).
Along with those few beneficial effects, consumption of areca nuts as a
masticatory substance is associated with the occurrence of many pathological
conditions. Previous studies have been demonstrated that AN chewers have higher
prevalence in periodontal diseases and oral, pharyngeal and esophageal cancers
where as in year 2003, the International Agency for Research on Cancer (IARC)
classified AN as a group 1 human carcinogen (IARC, 2004). Recently, it has been
3
also reported that AN chewing may increase the risk of systemic inflammation
(Shafiqueet al., 2012).
The content of alkaloid and flavonoid in AN plays a major role in the
pathogenesis particularly in oral submucous fibrosis by progressively enhancing
the collagen production, strengthening cross-links and reducing the degradation
(Prabhu et al., 2014). Also it is found in a study conducted using that areca nut
extracts have the ability to inhibit growth, attachment and matrix protein synthesis
of healthy human gingival fibroblasts (Chang et al., 1998). Moreover, there is
evidence that AN extracts have the ability to interfere with the antimicrobial
activity of neutrophils which might alter the microbial ecology of the oral cavity
promoting the bacterial colonization and periodontitis (Hang et al., 2000). In
addition, AN extract has shown an antimicrobial property against some oral
microorganisms including Streptococcus mutans, Streptococcus salivarius,
Fusobacterium nucleatum and Candida albicans which was affected least (de
Miranda et al., 1996). Furthermore, it is found that there is a higher proportion of
Helicobacter pylori colonization in patients with periodontitis (Gebara et al., 2004)
and a high prevalence of H. pylori in betel chewers compared to non-betel
chewers (Fernando et al., 2009).
H. pylori is a small, spiral, highly motile, microaerophilic gram-negative
bacterium found in human stomach. Though human is the principal reservoir, H.
pylori infection can be spread via water or undercooked vegetables contaminated
4
with sewage (Feldman et al., 1998; Dube et al., 2009). Faeco-oral, gastric-oral
and oral-oral transmissions are known to be the possible routes of transmitting the
infection (Brown, 2000).
H. pylori have many virulence factors such as vacA, cagA, and iceA which
revealed the bacterial relationship between the gastric mucosal surface and
causing disease. A complex of protein called vacuolating cytotoxin, VacA forms
acidic vacuoles in the host epithelial cells which leads to the cell death (Tanih et
al., 2010). The group of proteins called IceA has a homology to type II restriction
endonuclease and the IceA expression is upregulated with the contact between the
bacterium and host epithelial cells (van Doorn et al., 1999; Arents et al., 2001).
The cytotoxin-associated gene A (CagA) is a well characterized virulent
determinant of H. pylori. It is an oncoprotein which is injected into gastric
epithelial cells through the type IV secretion system. CagA can interfere and
unsettle the multiple host signaling pathways. This creates a direct oncogenic
insult which leads to the development of gastric cancer (Hatakeyama, 2014).
Furthermore, H. pylori produce enzymes including urease in order to evade the
harsh acidic environment in the stomach. Urease converts urea into ammonia and
hydrogen carbonate thus neutralizing the acidic pH in the cytoplasm and on the
periplasm (Tanahashi et al., 2000). Together with all of these virulence properties,
infection of H. pylori creates a noticeable inflammatory response with the
infiltration of various immune cells into the infected gastric mucosa leading to
5
damage (Tanih et al., 2010).
H. pylori is the major causative agent for chronic gastritis and peptic ulcer
disease as well as a cause of gastric cancers (IARC, 1994). Association between H.
pylori infection and gastric disease development is also found in studies using
animal models (Fox et al., 1995; Li et al., 1998; Franco et al., 2005; Arnold et al.,
2011). Among those models, mice model using strain PMSS1 is widely used
(Arnold et al., 2011; Krakowiak et al., 2015; Serizawa et al., 2015). However,
development of gastric cancer in experimental animals is rarely observed. To
promote the development of gastric cancer in experimental animals, chemical
carcinogen such as N-methyl-N-nitrosourea (MNU) is used in animal experiments
(Fort et al., 1974; Fujita et al., 1975).
In addition, Fernando et al. suggested that the chewing of betel quid containing
AN, lime, with or without tobacco may predispose H. pylori-infected one to the
development of severe gastric diseases including gastric cancers during chewing
and swallowing (Fernando et al., 2009). Thus, to identify association between AN
intake and H. pylori-induced gastric disease, we investigated effects of AN on
gastric disease lesion and H. pylori colonization of H. pylori PMSS1 strain-
infected mice.
Even though the AN intake is strongly associated with the occurrence of oral,
pharyngeal, and esophageal cancer as well as systemic inflammation, the
association between AN intake and development of gastric lesion has not been
6
identified yet. The aim of this study is to investigate the effect of AN on gastric
diseases using a mouse model for H. pylori infection.
7
II. MATERIALS AND METHODS
1. Diet and water preparation
Dry areca nut was finely powdered and mixed with AIN76A diet (Research Diets
Inc., New Brunswick, NJ, USA) at concentration of 2.5% (w/w). The diet
containing areca nut was pelleted, sealed in air-tight vinyl bags, and stored at 4 °C
until use. MNU was purchased from Sigma-Aldrich (St. Louis, MO, USA) and
dissolved in drinking water at concentration of 200 ppm.
2. Animal experiments
Five-week-old male C57BL/6 mice were purchased from Orient-Bio Inc. (Seoul,
Korea). The animals were maintained in a temperature-controlled room at 22 °C
with a 12-h light-dark cycle. Food and water were provided ad libitum. After one
week of acclimation, mice were put into experiment. The first experiment was
conducted as a preliminary study, to examine the ability of H. pylori PMSS1
strain to induce gastric diseases in mice, and to examine the short-term and long-
term effect of H. pylori infection in mice. To examine the short-term and long-
term effect of H. pylori infection, experiments were for 4 weeks and 12 weeks,
respectively. Mice were divided into 2 groups: control (n = 2 for 4-week and n = 3
8
for 12-week experiment) and PMSS1-infected group (n = 3 for both experiments).
We inoculated mice with H. pylori PMSS1 strain and brucella broth for PMSS1-
infected group and control group, respectively.
In the second experiment, mice were divided into seven groups: ND (n = 6), AD
(n = 8), MNU (n = 8), ND/HP (n = 10), AD/HP (n = 10), MNU/HP (n = 8), and
AD/MNU/HP (n = 6) (Figure 1). After intragastric inoculation with H. pylori
PMSS1 strain at 6 weeks, ND/HP and ND/HP/MNU mice were fed with AIN76A
diet for 10 weeks. In contrast, AD/HP and AD/HP/MNU mice were fed AIN76A
diet containing 2.5% AN for 10 weeks. As a negative control, ND mice that were
administered with brucella broth were fed with the AIN76A diet. AD mice that
were administered with brucella broth were fed with the AIN76A diet. Body
weight and food intake were measured every week during the experimental period.
At the end of each experimental period, mice were sacrificed, gastric mucosal
tissues were weighed and then examined histologically, and H. pylori colonization
was determined as described below. The animal experiments were performed in
accordance with institutional guidelines. Protocols were approved by the animal
ethics committee of the Yonsei University College of Dentistry (2012-0076).
9
10
Figure 1. Experimental design. The mice of ND group were inoculated with
brucella broth (▽) and fed with normal diet ( ). AD group mice were inoculated
with brucella broth and fed with 2.5% AN-containing diet ( ). MNU group mice
were inoculated with brucella broth and fed with 200 ppm MNU-containing water
( ). ND/HP group mice were inoculated with H. pylori strain PMSS1 (▼) and
fed with normal diet. AD/HP group mice were inoculated with H. pylori strain
PMSS1 and fed 2.5% AN-containing diet. MNU/HP group mice were inoculated
with H. pylori strain PMSS1 and fed with 200 ppm MNU-containing water.
AD/MNU/HP group mice were inoculated with H. pylori strain PMSS1 and fed
with 2.5% AN-containing diet and 200 ppm MNU-containing water.
11
3. H. pylori culture and mouse infection
H. pylori PMSS1 strain was stored as frozen stock at -80 °C in brain-heart
infusion medium supplemented with 20% glycerol and 10% fetal bovine serum
(FBS) (Gibco-BRL, Grand Island, NY, USA). H. pylori was grown on horse blood
agar plates containing 4% Columbia blood agar base (Oxoid, Basingstoke,
Hampshire, UK), 5% defibrinated horse blood (HemoStat Labs, Dixon, CA, USA),
0.2% b-cyclodextrin, 10 mg/mL vancomycin, 2.5 U/mL polymyxin B, 5 mg/mL
trimethoprim, and 8 mg/mL amphotericin B at 37 °C under microaerobic
condition. A microaerobic atmosphere was generated using a CampyGen sachet
(Oxoid) in a gas-pak jar (BD, Franklin Lakes, NJ, USA). For liquid culture, H.
pylori was grown in brucella broth (Difco & BBL Diagnostics, Franklin Lakes, NJ,
USA) containing 10% FBS and 10 μg/mL vancomycin. Cultures were shaken at
110 rpm, in a microaerobic environment. According to the growth curve, 2 × 107
bacteria were collected and resuspended in 250 μL of brucella broth, which was
inoculated for mouse infection via intragastric gavage after fasting for 24 h.
4. Determination of the number of viable H. pylori in the stomach
The number of viable H. pylori in the mice stomach was determined as
previously described with minor modifications (Takahashi et al., 1998). After the
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mice were fasted for 18 hr, they were euthanized, and their stomachs were excised.
The stomach was dissected along the greater curvature, dissected to remove
forestomach, mildly washed with phosphate-buffered saline (pH 7.4) to remove
food debris, and then divided longitudinally into two halves. One half of each
stomach was homogenized in 1 mL of brucella broth using a homogenizer
(Kinematica, Luzern, Switzerland). The diluted homogenates were applied to
horse blood agar plates used to culture H. pylori, additionally supplemented with
100 μg/mL vancomycin, 100 μg/mL bacitracin, and 10 μg/mL nalidixic acid. The
plates were incubated at 37 °C under microaerobic condition for 5 days. The
colonies were counted and the number of viable H. pylori was expressed as
colony forming units (CFU) per gram of stomach tissue.
5. Histological analysis
The other half of each stomach was fixed in 10% neutral buffered formalin and
embedded in paraffin. Paraffin sections were cut into 4 mm and stained with
hematoxylin and eosin for morphological observation. Two aspects of gastric
lesions were recorded according to the updated Sydney system:
polymorphonuclear leukocytes (PMNs) infiltration and chronic inflammation as
lymphocyte infiltration. Morphological features of the gastric antrum and corpus
were graded on a 0 to 4 scale: Grade 0, normal; Grade 1, slight; Grade 2, mild;
13
Grade 3, moderate; and Grade 4, severe (Dixon et al., 1996). Microscopic images
were obtained at magnification of × 100 for corpus and magnification of × 200 for
antrum.
6. Statistical analysis
The SPSS statistics 23 program (IBM Corp., Armonk, NY, USA) was utilized for
all statistical analyses. An analysis of variance was used to compare different
groups, and multiple comparison was performed using Scheffe’s method. A P-
value of < 0.05 was regarded as statistically significant.
14
III. RESULTS
1. Establishment of mouse model for H. pylori-induced gastric disease
development
To examine the ability of H. pylori PMSS1 strain to induce gastric diseases in
mice, we inoculated mice with H. pylori PMSS1 strain. After 4 weeks and 12
weeks from inoculation, mice were sacrificed, number of viable H. pylori was
measured and pathologic change of stomach tissue was graded. H. pylori PMSS1
strain successfully colonized mice stomach. In average, 1.1 × 104 bacterial cells
were recovered from mice infected for 4 weeks. After 12 weeks from infection,
3.5 × 104 bacterial cells, approximately 3 times more bacterial cells were
recovered than those recovered from mice infected for 4weeks (Figure 2).
Infiltration of lymphocytes and PMNs were graded in antrum and corpus of
stomach. Grades of lymphocytes and PMNs infiltration were appeared similarly in
both antrum and corpus. After 4 weeks from infection, slight lymphocytes and
PMNs infiltration was appeared in mice stomach. After 12 weeks from infection,
inflammation was aggravated. Stomachs showed mild to moderate lymphocytes
and PMNs infiltration after 12 weeks from infection (Figure 3).
15
Figure 2. Colonization of H. pylori PMSS1 in mice stomach after 4 weeks and 12
weeks from infection.
16
17
18
Figure 3. Histopathological evaluation of mice stomach after H. pylori infection.
Infiltration of PMNs in corpus and antrum, infiltration of lymphocytes in corpus
and antrum were graded after 4 weeks (●) and 12 weeks (○) from infection,
according to the updated Sydney system.
19
2. Gross changes of food intake and body weight of mice
To examine whether AN and MNU feeding caused gross changes of food intake
and body weight of mice, we measured food intake and body weight for 10 weeks.
During the period, the food intake of all groups were similar (Fig. 4A). In the
changes of body weight gain of mice, that of AD group was increased, compared
to MNU, ND/HP, MNU/HP, and AD/MNU/HP groups (Fig. 4B). But, importantly,
all groups except AD group, showed no difference for the food intake and the
body weight gain.
20
Fig
ure
4.
Gro
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hang
es o
f fo
od i
ntak
e an
d bo
dy w
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t in
mic
e du
ring
the
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. (A
) F
ood
inta
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(B)
Bod
y w
eigh
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ange
. D
ata
are
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SD
. *
indi
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.
21
3. Effect of AN and MNU on viable H. pylori colonization in the H. pylori-
infected mouse stomach
To determine the effect of AN and MNU on H. pylori colonization in gastric
mucosa of mice, the number of H. Pylori was measured at 10-week after normal
diet, AN-containing diet, and MNU-containing water feeding. Obviously, H.
pylori-infected groups such as ND/HP, AD/HP, MNU/HP, and AD/MNU/HP
groups showed significant increase in H. pylori colonization compared to non-
infected groups (Fig. 5A). However, there was no significant difference in H.
pylori colonization among all groups infected with H. pylori, suggesting that
administration of AN and MNU didn’t affect H. pylori colonization.
4. Effect of AN and MNU on the stomach weight of H. pylori-infected mice.
To determine the effect of AN and MNU on the stomach weight of H. pylori-
infected mice, the stomach weight was measured at 10-week after normal diet,
AN-containing diet, or MNU-containing water feeding and was expressed as the
ratio of stomach weight to body weight. The stomach weight of all four groups
infected with H. Pylori were significantly increased, compared to non-infected
groups (Fig. 5B). However, there was no significant difference among all H.
pylori-infected groups, suggesting that AN or MNU treatment didn’t affect gross
stomach weight.
22
23
24
Figure 5. The viable number of H. pylori colonization in the gastric mucosa and
the ratio of stomach weight to body weight in H. pylori-infected mice. (A) The
viable number of H. pylori colonization in the gastric mucosa was evaluated using
a whole stomach culture system. (B) The stomach weight was measured at 10-
week after normal or AN-containing diet feeding and was expressed as the ratio of
stomach weight to body weight in H. pylori-infected mice. * indicates a
significant difference at P<0.05.
25
5. Effect of AN and MNU on gastric lesions of H. pylori-infected mice
To determine whether AN and MNU affects H. pylori-induced gastric lesion, we
observed the histopathological changes such as infiltration of PMNs and
mononuclear cells in corpus and antrum parts of stomach (Fig. 6A). Although
PMNs infiltration in all groups did not show any difference, infiltration of
lymphocytes in H. pylori-infected groups (ND/HP, AD/HP, MNU/HP, and
AD/MNU/HP) were significantly increased compared to those in non-infected
groups (Fig. 6B and 6C). More importantly, infiltration of lymphocytes in AD/HP
group was more severe than that in ND/HP group (Fig. 6C). Additionally, it is
worth to note that no hyperplastic or metaplastic lesions were observed in any
group throughout experimental period.
26
A
27
28
29
Figure 6. Effect of AN and MNU on H. pylori-induced gastric lesions of mice. (A,
upper panel) Corpus part of gastric mucosa was stained with hematoxylin and
eosin. Microscopic images were obtained at magnification ×100. (A, lower panel)
Antrum part of gastric mucosa was stained with hematoxylin and eosin.
Microscopic images were obtained at magnification ×200. (B) Histological
grading of the PMNs infiltration in gastric mucosa. (C) Histological grading of the
lymphocyte infiltration in gastric mucosa. The inflammatory responses of gastric
mucosa were graded according to morphologic criteria. * indicates a significant
difference at P < 0.05.
30
IV. DISCUSSION
The distinctive characteristic of H. pylori infection is chronic inflammation of
stomach (Philpott et al., 2002). Various animals were exploited to develop
experimental animal models for investigating human H. pylori infection. Recently,
it has been reported that certain H. pylori strains were able to colonize the gastric
mucosa of mice or Mongolian gerbils (Marchetti et al., 1995; Watanabe et al.,
1998). Among several animal models with H. pylori infection, it has been known
that mice infected with H. pylori appeared mild responses of gastric inflammation
(Ferrero et al., 1998). The commonly used mouse-adapted strain was H. pylori
SS1, which does not translocate a virulence factor, cytotoxin-associated gene A
(CagA) into the host cells nor induce interleukin (IL)-8 (Crabtree et al., 2002).
Since possession of the functional CagA in H. pylori strains is associated with
more severe gastric inflammation and development of neoplastic gastric disease
(Blaser et al., 1995; Philpott et al., 2002), H. pylori PMSS1 strain with a
functional CagA and ability to translocate CagA to host cells has been employed
for the mouse model (Arnold et al., 2011). In this study, we found that H. pylori
strain PMSS1 induced moderate chronic inflammation in gastric mucosa of mice.
This suggests that the mice model infected with H. pylori PMSS1 strain probably
mimics the human’s asymptomatic response to CagA-positive H. pylori infection.
Furthermore, the preneoplastic lesions such as glandular hyperplasia and
31
metaplasia were not observed during the H. pylori infection period. To develop
preneoplastic lesions, it might be necessary to lengthen the duration of infection
or to treat chemical carcinogens in combination with H. pylori infection.
In mice without H. pylori infection, mice fed with 2.5% AN or 200 ppm MNU
showed no inflammatory changes compared with control ND group during the
experimental period. In contrast, in the presence of H. pylori infection, mice fed
with 2.5% AN showed more severe lymphocyte infiltration than ND/HP mice
group fed with normal diet in the presence of H. pylori infection. Previous studies
have reported that AN extracts increased expression of inflammatory cytokines
such as tumor necrosis factor-a, IL-1b, IL-6 and IL-8 (Chang et al., 2009), and H.
pylori induces increased production of proinflammatory cytokines (Harris et al.,
2000; Guiraldes et al., 2001). Combined with those findings, our data suggest that
AN treatment aggravates H. pylori-induced gastritis by inducing increased
expression of inflammatory cytokines in gastric mucosa. Considering that fewer
than 10% of H. pylori-infected patients showed symptomatic gastric diseases, it
can be assumed that AN may affect disease progression from asymptomatic
infection to severe gastric disease. Because gastric cancer development progresses
from H. pylori-induced gastritis (Peek and Blaser, 2002), intake of AN may affect
H. pylori-induced gastric carcinogenesis. Since there was no significant difference
in bacterial colonization between H. pylori-infected groups of ND/HP and AD/HP,
the more severe chronic gastric inflammation with more lymphocyte infiltration in
32
AD/HP was likely due to the effect of AN itself but not due to the difference of H.
pylori colonization. Further studies are necessary to find out what is the main
pathway related with the progression of gastric inflammation by AN intake.
In contrast to AN, treatment of 200 ppm MNU did not induced significant
changes, as there were no statistically significant difference in all parameters,
between ND/HP and MNU/HP groups, and between AN/HP and AN/MNU/HP
groups. However, MNU-treated groups (MNU/HP and AD/MNU/HP) showed
slightly increased lymphocyte infiltration, compared to corresponding groups
without MNU treatment (ND/HP and AD/HP) though the differences were not
statistically significant. The lack of significance might be due to low
concentration of MNU or short duration of H. pylori infection.
In addition, mice treated with MNU such as MNU/HP and AD/MNU/HP groups,
showed relatively low H. pylori colonization compared to non-MNU treated
groups such as ND/HP and AD/HP groups, though the differences were not
statistically significant. It is reported that H. pylori can colonize gastric mucosa,
but can poorly colonize atrophic mucosa and can hardly colonize intestinal
metaplasia at all (Blaser and Atherton, 2004). This may imply that MNU induces
histological changes which makes gastric environment unfavourable to H. pylori.
In conclusion, we confirmed availability of H. pylori strain PMSS1and C57BL/6
mice as a model system for H. pylori-induced gastric disease development. Using
this model, we found that AN induced more severe chronic gastritis in H. pylori
33
strain PMSS1 infection, and the chewing of betel quid containing AN may
predispose H. pylori-infected one to the development of severe gastric diseases
including gastric cancers. In addition, administration of MNU in H. pylori
PMSS1-infected mouse model showed possibility to be used as model system to
study H. pylori-induced gastric carcinogenesis.
34
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42
문 약
스에 Helicobacter pylori 에 해
염에 한 Areca nut 향
< 수 차 >
연 학 학원 학과
계 여러 역에 많 가 Areca nut 담 나 가루 함께
에 싸 씹어 태 하고 다. Areca nut ,
점 하 등 전암병 , 강암 등 강내 뿐만 아 라 염 , 비만,
당뇨, 저 압, 간 , , 순 등 전신 과 다.
Helicobacter pylori 50% 감염시키고 , 염, 양, 암과
같 키 병원균 다. 특히, 암 한 망 3 째 높다고
알 암과 연 문에 균 에 하게 1 암 어
다.
43
Areca nut 높 빈 H. pylori 높 감염 고 해 볼 , 강암
키 Areca nut 취가 H. pylori 에 한 생에 미 향에 한
연 가 필 하다. 라 본 연 스에 H. pylori 에 해 염에 한
Areca nut 향 알아보고 하 다. 실험 한 H. pylori 스
조 에 정착하 것 알 져 PMSS1 균주 하 다. 스들 7 개
그룹 하여 실험 시행하 다. 1 정 료 물 , 2 Areca
nut 포함 료 물 , 3 정 료 N-methyl-N-nitrosourea(MNU)
함 한 물 취시켰다. 4 H. pylori 감염시킨 정 료 물 , 5
H. pylori 감염시킨 Areca nut 포함 료 물 , 6 H.
pylori 감염시킨 정 료 MNU 가 함 물 , 7 째 실험 H. pylori
감염시킨 Areca nut 포함 료 MNU 가 함 물 취하 하 다.
10 주간 실험 간 동안, 료 취량과 체 각 집단 에 한
차 나타내 않았고 집락 수 무게 H. pylori 가 감염 들에
가하 다. H. pylori 집락 수가 가한 것 실험에 H. pylori 가 스 에
공적 감염 었 나타낸다. 10 주 조 학적 검 에 H. pylori 가
감염 들에 보다 프 많 나타났다. 특히, H. pylori 가
감염 들 Areca nut 포함 료 취한 료 취한 에
비해 심한 프 양 보 다. Areca nut H. pylori 에 해
만 염 악 시키 것 해 할 수 나타낸다. 실험에
MNU 처 여 실험 결과에 한 향 미 않았 , Areca nut 암
가 알아보 해 본 연 에 한 스 에 다양한 MNU
농 처 하여 적 찰할 필 가 다.
심 말: Areca nut, Helicobacter pylori, 염, 강암