Analysis of Helicobacter pylori and nonsteroidalanti-inflammatory drug-induced gastric epithelial injury

M. IGARASHI, A. TAKAGI, X. JIANG*, K. HASUMI, S. WATANABE, R. DEGUCHI & T. MIWA

Division of Gastroenterology, Department of Internal Medicine, Tokai University School of Medicine, Kanagawa, Japan;

and *Beijing University, Beijing, China

INTRODUCTION

Helicobacter pylori is a Gram-negative bacterium that has

been recognized as a causative agent for gastritis and

gastroduodenal ulcer.1–3 H. pylori has also been implica-

ted in the pathogenesis of gastric cancer and lymphoma.4

Moreover, H. pylori and nonsteroidal anti-inflammatory

drugs (NSAIDs) account for nearly all gastroduodenal

ulcers and serious ulcer complications. However, the

relationship between H. pylori and NSAID-related gas-

troduodenal mucosal injury has not been clarified.

Most drugs that assist ulcer healing stimulate

mucus production and suppress acid and pepsinogen

secretion.5–7 Sofalcone, 2-carboxy-methoxy-4,4-bis(3-

methyl-2-butenyloxy)chalone, one of the sophoradin

derivatives, has long been used in traditional Chinese

medicine for the treatment of digestive diseases. It has

been reported that sofalcone has a cytoprotective exerts

ulcer healing effects.8 To determine the role of H. pylori

in NSAID-induced gastric mucosal injury, we examined

these factors in gastric mucosal injury using gastric

epithelial cells. In addition, we studied the effect of

sofalcone, a gastric mucosal protection agent, on

H. pylori and NSAID-induced gastric mucosal injury.

MATERIALS AND METHODS

Bacterial strains and their characterization

Two clinical strains of H. pylori were isolated from

gastric biopsy specimens from patients with gastric and

duodenal ulcer at Tokai University Hospital. The

SUMMARY

Background: Helicobacter pylori and nonsteroidal anti-

inflammatory drugs (NSAIDs) are important factors in

gastric mucosal injury. However, the relationship

between H. pylori and NSAID-related gastroduodenal

mucosal injury has not been clarified.

Aim: To determine the role of H. pylori in NSAID-

induced gastric mucosal injury and to examine the

effects of H. pylori, indomethacin and sofalcone on

gastric epithelial cells in culture, as a useful model to

study gastric mucosal injury. In addition, we studied the

effect of sofalcone, a gastric mucosal protection agent, on

H. pylori and NSAID-induced gastric mucosal injury.

Methods: Cytotoxic and noncytotoxic strains of H. pylori

were used, each with an inoculum of 107 cfu/mL. The

effect on the growth of RGM–1 cells (a rat gastric

epithelial cell line) was studied by MTT assay, and levels

of prostaglandin E2 in culture supernatants were

measured by EIA.

Results: Both cytotoxic and noncytotoxic strains of

H. pylori tended to induce cell injury in RGM-1 cells at

48 h after inoculation. Indomethacin alone induced

gastric epithelial injury in a dose-dependent manner, but

did not augment cell injury induced by H. pylori. In

addition, sofalcone (10)5 mol/L) showed a suppressive

effect on indomethacin-induced gastric epithelial injury.

Conclusion: These findings indicate that indomethacin

induces gastric mucosal injury regardless of H. pylori

infection, and suggests that sofalcone may be a use-

ful drug in the treatment of NSAID-induced mucosal

injury.

Ó 2002 Blackwell Science Ltd 235

Correspondence to: Dr M. Igarashi, Division of Gastroenterology, Depart-

ment of Internal Medicine, Tokai University School of Medicine, Isehara,Kanagawa, Japan.

Aliment Pharmacol Ther 2002; 16 (Suppl. 2): 235–239.

bacteria were grown on blood agar plates as previously

described.9 The presence of vacuolating cytotoxin (VT)

was assayed by cytotoxic assay using RK 13 cells.10

Hp69 was VT-positive, and Hp78 was VT-negative.

H. pylori was resuspended into D-MEM/F12 with 20%

heat-inactivated foetal bovine serum (FBS).

Cell culture

The rat gastric epithelial cell line RGM-1 was obtained

from the Japanese Cancer Research Resources Bank

(Tsukuba, Japan). RGM-1 cells were grown in D-MEM/

F-12 (Life Technologies, Rockville, MD, USA) containing

20% heat-inactivated FBS (Life Technologies). RGM-1

cells were seeded in 96-well flat-bottom microtitre

plates, at a density of 104/well for the MTT assay and

103/well for the PGE2 assay.

MTT assay

A cell proliferation kit (Boehringer Mannheim, Mann-

heim, Germany) was used to measure cytotoxicity

according to the manufacturer’s instructions. Indometh-

acin was dissolved in DMSO (1 mL). H. pylori was added to

the microtitre plates, followed by indomethacin. In some

experiments, sofalcone was also added. Briefly, after 48 h

of incubation, 10 lL of MTT labelling reagent (final

concentration 0.5 mg/mL) was added to each well. The

microtitre plate was incubated for 4 h in a CO2 incubator.

A solubilization solution (100 lL) was added to each well,

and the plate was kept overnight in a CO2 incubator. The

spectrophotometric absorbance of the sample was meas-

ured at 550 nm. The reference wavelength was 650 nm

for prostaglandin E2, which was measured with a

prostaglandin E2 enzyme immunoassay (EIA) system

(Amersham Pharmacia Biotech, Amersham, Bucks, UK).

Statistical analysis

Comparisons were performed by a one-way factorial

ANOVA and multiple-comparison tests. P < 0.05 was

considered statistically significant.

RESULTS

Cell growth

Both cytotoxic (Hp69) and noncytotoxic (Hp78) strains

of H. pylori tended to induce cell injury in RGM-1 cells at

48 h. As shown in Figure 1, the level of cell injury was

not significantly different between RGM-cells and Hp69

(P ¼ 0.331), Hp78 (P ¼ 0.09). Indomethacin induced

cell injury in RGM-1 cells at 48 h in a dose-dependent

manner. Cell viability (%) of indomethacin (400 lmol/L

and 500 lmol/L was 80% (n.s.), and 50% (P ¼ 0.001),

respectively (Figure 2). However, combinations of indo-

methacin with H. pylori did not augment cell injury in

RGM-1 cells at 48 h (Figure 3). Sofalcone at a concen-

tration of 10)5 mol/L did not significantly prevent

indomethacin-induced cell injury (Figure 4).

100

90

80

70

60

50

40

30

20

10

0

Cel

l via

bilit

y (%

)

Cont H.p 69 H.p 78

P = 0.331 P = 0.09

Figure 1. Effect of Helicobacter pylori on

cell injury in RGM-1 cells (48 h) after

inoculation. Data are mean ± S.D.

(n ¼ 8).

236 M. IGARASHI et al.

Ó 2002 Blackwell Science Ltd, Aliment Pharmacol Ther 16 (Suppl. 2), 235–239

Prostaglandin E2 production

As shown in Table 1, indomethacin (400–500 lmol/L)

decreased prostaglandin E2 level in culture supernatant

in a dose- dependent manner. Further, sofalcone (10–4–

10–5 ) reversed the decrease in levels of prostaglandin E2

by indomethacin.

DISCUSSION

H. pylori and NSAIDs are important factors involved in

gastric mucosal injury. However, the relationship

between H. pylori and NSAID-related gastroduodenal

mucosal injury has not been clarified. A putative

relationship was suggested because H. pylori infection

is the cause of chronic active gastritis, and polymor-

phonuclear leukocytes may be important mediators of

NSAID-related mucosal injury.11 However, conflicting

findings have been reported regarding H. pylori eradi-

cation therapy in NSAID users. Chan et al.12 reported

that eradication of H. pylori before NSAID therapy

reduced the occurrence of NSAID-induced peptic ulcers.

However, Hawkey et al.13 reported that H. pylori

eradication in long-term users of NSAIDs with peptic

ulcers led to impaired healing of gastic ulcers and did

not affect the rate incidence of peptic ulcers.

Furthermore, COX-2 expression has been reported in

H. pylori-infected mucosa, suggesting that H. pylori

100

90

80

70

60

50

40

30

20

10

0

Cel

l via

bilit

y (%

)

Cont 400 µmol/L 500 µmol/L

N.S. P<0.001

Figure 2. Effect of indomethacin on cell

injury in RGM-1 cells (48h) after inocu-

lation. Data are mean ± S.D. (n ¼ 8).

140

120

100

80

60

40

20

0

Cel

l via

bilit

y (%

)

Cont 300 µmo/L 400 µmo/L 500 µmo/L300 µmo/L+H.p 78

400 µmo/L+H.p 78

H.p 78

P<0.001

Figure 3. Effect of Helicobacter pylori

(H.p 78) and indomethacin on cell injury

in RGM-1 cells. Data are mean ± S.D.

(n ¼ 8).

ANALYSIS OF H. PYLORI AND NSAID-INDUCED GI INJURY 237

Ó 2002 Blackwell Science Ltd, Aliment Pharmacol Ther 16 (Suppl. 2), 235–239

infection may reduce NSAID-associated gastric mucosal

injury.14 Cell proliferation and apoptosis are essential

events in the cellular turnover of gastric tissue. Gastric

cancer cell lines have been used for investigation of

H. pylori-induced apoptosis.15

Previously, we reported that H. pylori did not induce

cell death alone but did induce cell death in the presence

of interferon-c and TNF-a.9 In in vitro studies, gastric

cancer cell lines have been used to clarify relationship

between H. pylori and epithelial cell, however, the

cancer cell lines seem to be unsuitable for the study of

NSAIDs. In the present study, we used RGM-1 cells from

normal rat stomach and showed that cytotoxic and

noncytotoxic strains of H. pylori tended to cause or

induce cell injury in RGM-1 cells at 48 h.

Furthermore, we confirmed that indomethacin dose-

dependently induced cell injury in RGM-1 cells at 48 h.

In addition, indomethacin did not augment the cell

injury induced by H. pylori. This might have occurred

because the experiments were done with an in vitro

culture system, where normal inflammatory mediators

that accompany infection and which might be import-

ant for the combined effects of H. pylori and NSAIDs are

not present. The mechanism by prevented indometha-

cin from augmenting the cell injury induced by H. pylori

in RGM-1 remains unknown. However, H. pylori and

indomethacin may be independent factors involved in

gastric mucosal injury.

Romano et al.16 reported that H. pylori up-regu-

lates COX-2 mRNA expression and prostaglandin E2

synthesis in MKN 28 cells. Sofalcone exerts its

cytoprotective and ulcer healing effect by various

mechanisms. Slomiany et al.17 reported that sofalcone

increases mucous gel thickness, and that the content

of sulfo- and sialo-mucins in the gel increased by 54

and 25, respectively. There was also a substantial

increase in mucous glycoprotein.18 A sofalcone-

induced increase in prostaglandin secretion has also

been reported, which is significant because certain

prostaglandins inhibit histamine- or gastrin-stimulated

gastric acid secretion.19–21 Robert et al. reported that

prostaglandins protect the gastric mucosa against

necrotizing agents against lipid peroxidation mediated

by oxygen radicals.

Sofalcone can also block the action of these toxic agents

and their induction of gastric mucosal injury.22 In

the present study, we confirmed that NSAIDs (400–

500 lmol/L) decreased prostaglandin E2 level in culture

Table 1. Effect of indomethacin on prostaglandin E2 production

in RGM)1 cells. Data are the mean of three experiments

Prostaglandin

E2 (pg/well)

Control 275.2 ± 74.5

Indomethacin (400 lM) 292.6 ± 60.1

Indomethacin (500 lM) 174.1 ± 12.7

Indomethacin (500 lM) + sof. 10)4 262.1 ± 66.0*

Indomethacin (500 lM) + sof. 10)5 260.9 ± 140.5

Data are the mean of three experiments. *P < 0.05: indomethacin

vs. indomethacin + sofalcone 10)4 mol/L.

Cont 400 µmol/L 400 µmol/L+sof. 10–5

100

90

80

70

60

50

40

30

20

10

0

Cel

l via

bilit

y (%

)

N.S.

Figure 4. Effect of sofalcone on gastric

epithelial injury induced by indometha-

cin. Data are mean ± S.D. (n ¼ 8).

238 M. IGARASHI et al.

Ó 2002 Blackwell Science Ltd, Aliment Pharmacol Ther 16 (Suppl. 2), 235–239

supernatant in a dose-dependent manner, and that

sofalcone (10)4)10)5 mol/L) suppressed this decrease

in prostaglandin E2 level. Sofalcone at a concentration

of 10–5 mol/L did not significantly prevent indo-

methacin-induced cell injury. In contrast, sofalcone

(10)4)10)5 mol/L) reversed the decrease in levels of

prostaglandin E2 by indomethacin.

Although prevention of indomethacin-induced cell

injury by sofalcone was not observed in vitro, sofalcone

seems to be effective for the treatment of NSAID-

induced cell injury, by activating prostaglandin

production in vivo.

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