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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

１２

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

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ure

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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