Volume 9 Number 2 2004 H E L I C O B AC T E R

Teprenone, but not H2-Receptor Blocker or Sucralfate, Suppresses Corpus Helicobacter pylori Colonization and Gastritis in Humans: Teprenone Inhibition of H. pylori-Induced Interleukin-8 in MKN28 Gastric Epithelial Cell Lines
Blackwell Publishing, Ltd.

Kazumasa Miyake,* Taku Tsukui,* Yoko Shinji,* Kei Shinoki,* Tetsuro Hiratsuka,* Hitoshi Nishigaki,* Seiji Futagami,* Ken Wada,* Katya Gudis,* Katsuhiko Iwakiri,* Nobutaka Yamada and Choitsu Sakamoto*
* Third

Department of Internal Medicine, Department of Pathology; Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo, Japan

ABSTRACT

Background. The role of teprenone in Helicobacter pylori-associated gastritis has yet to be determined. To investigate the effect of teprenone on inammatory cell inltration, and on H. pylori colonization of the gastric mucosa in H. pylori-infected patients, we rst compared the effect of teprenone with that of both histamine H2 receptor antagonists (H2-RA) and sucralfate on the histological scores of H. pylori gastritis. We then examined its in vitro effect on H. pyloriinduced interleukin (IL)-8 production in MKN28 gastric epithelial cells. Materials and Methods. A total of 68 patients were divided into three groups, each group undergoing a 3month treatment with either teprenone (150 mg/day), H2-RA (nizatidine, 300 mg/day), or sucralfate (3 g/day). All subjects underwent endoscopic examination of the stomach before and after treatment. IL-8 production

in MKN28 gastric epithelial cells was measured by enzyme-linked immunosorbent assay (ELISA). Results. Following treatment, the teprenone group showed a signicant decrease in both neutrophil inltration and H. pylori density of the corpus (before vs. after: 2.49 0.22 vs. 2.15 0.23, p = .009; 2.36 0.25 vs. 2.00 0.24, p = .035, respectively), with no signicant differences seen in either the sucralfate or H2-RA groups. Teprenone inhibited H. pylori-enhanced IL-8 production in MKN28 gastric epithelial cells in vitro, in a dose-dependent manner. Conclusions. Teprenone may modify corpus H. pyloriassociated gastritis through its effect on neutrophil inltration and H. pylori density, in part by its inhibition of IL-8 production in the gastric mucosa. Keywords. Teprenone, Helicobacter pylori, neutrophil inltration, interleukin-8, histamine H2 receptor antagonists, sucralfate.

olonization by Helicobacter pylori of the gastric mucosa results in chronic gastritis, which is characterized by abundant neutrophil inltration into the gastric mucosa accompanied by lymphocyte and macrophage inltration [1,2]. This infection-induced neutrophil inltration plays an important role in gastric epithelial injury and the development of peptic ulcer, and may also be associated with the progression to premalignant conditions such as atrophy, intestinal metaplasia and hypochlorhydria [25]. Various cytokines, such as interleukin (IL)-1, -6 and -8 and tumor necrosis factor (TNF)-, are associated

Reprint requests to: Kazumasa Miyake, Third Department of Internal Medicine, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan.
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with the pathogenesis of gastritis induced by H. pylori infection [68]. Among such cytokines, IL-8 induced in gastric epithelial cells infected with H. pylori [6,9] is a potent chemoattractant and activator of neutrophils [10,11]. Teprenone is a popular drug in Japan and is widely used for patients with peptic ulcer and gastritis, whether or not infected with H. pylori [12,13]. The mechanism by which teprenone exerts its mucoprotective effects has been thought to be the induction of heat shock protein and the stimulation of mucin secretion in the gastric mucosa [14,15]. However, it has yet to be determined whether teprenone has more specic actions against H. pylori-associated gastritis. Therefore, in the present study, to investigate the effect of teprenone on inammatory cell 130

Effects of Teprenone on H. pylori Gastritis inltration and H. pylori colonization in the gastritis mucosa of H. pylori-infected patients, we rst compared the effect of teprenone with that of histamine H2-receptor antagonists (H2-RA) and sucralfate on histological scores of gastritis with H. pylori infection, following a 3-month treatment. Then we focused on its in vitro effects on the production of IL-8, a key cytokine involved in neutrophil inltration in the stomach and induced by H. pylori infection in gastric epithelial cells.
Methods

131 more than 50 indicates their presence. Serum pepsinogen I and II levels were measured by a specic radioimmunoassay (RIA) (Dinabot, Tokyo, Japan). Serum gastrin levels were measured by a specic RIA using a gastrin RIA kit (Dinabot). Histology At each endoscopic examination, biopsy specimens from the antrum and corpus were obtained for histological analysis. Antrum biopsy specimens were taken within 2 cm of the pylorus, and corpus specimens from the mid-portion of the greater and lesser curvatures. We excised one or two biopsy specimens from each region, with enough gastric mucosa to sufce for study of the number of intraepithelial lymphocytes, severity of gastritis, and occurrence of H. pylori. The specimens were xed in 10% buffered formalin, embedded in parafn, cut into sections, and stained with hematoxylin-eosin for histological analysis and with Giemsa stain for H. pylori detection. Degree of inammation, activity and H. pylori density were assessed according to the modied updated version of the Sydney system and classied into four categories as follows: 0, none; 1, mild; 2, moderate; 3, severe [16]. The mean value of scores from greater and lesser curvatures was used as a histological score for the corpus. A histopathologist, blind to the clinical data for the patients, examined the biopsy specimens. Bacterial Strains and Culture Conditions H. pylori strain NCTC 11637 was obtained from the American Type Culture Collection (Rockville, MD). H. pylori was cultured on brain-heart infusion (BHI) agar plates supplemented with 5% horse blood for 24 hours under microaerophilic conditions at 37 C. The bacteria were harvested from the broth culture by centrifugation and resuspended at the indicated concentration in antibiotic-free RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS). Gastric Cancer Cell Line

Subjects Eighty-nine patients with dyspepsia or peptic ulcer scars were invited to participate in the present prospective, open, randomized study. H. pylori infection before eradication was conrmed in all patients by rapid urease test, histological examination of antral and corpus biopsy specimens, and measurement of serum antiH. pylori immunoglobulin G (IgG) antibodies. Patients were dened as H. pylori-positive if two or more of these tests were positive. We excluded patients without H. pylori infection, those with open peptic ulcers, previous gastrectomy, or malignant diseases, those who were pregnant, and those aged less than 16 years or more than 75 years. Patients who were taking nonsteroidal anti-inammatory drugs (NSAIDs), corticosteroids, antibiotics, proton pump inhibitors (PPIs) or H2-RA 4 weeks before initial endoscopy were also excluded. Patients who met all inclusion criteria gave informed consent before participation. We excluded 11 patients who were lost to follow-up or needed other treatment within 3 months. A total of 68 patients were divided into three groups: a teprenone group (150 mg/day, n = 23), an H2-RA group (nizatidine, 300 mg/day, n = 23), and a sucralfate group (3 g/day, n = 22); all were treated for 3 months. Endoscopic features of the stomach were evaluated before and 3 months after treatment. Laboratory Analysis Serum anti-H. pylori IgG was measured by enzyme-linked immunosorbent assay (ELISA) (Eiken, Tokyo, Japan). In this assay, an antibody ELISA value of less than 30 indicates the absence of detectable IgG antibodies, whereas a value of
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The human gastric cancer cell line MKN28 was obtained from the Japanese Cancer Research Resources Bank. MKN28 cells were grown in RPMI-1640 containing 10% heat-inactivated bovine calf serum, 100 units/ml penicillin G, and 100 mg/ml streptomycin.

132 Determination of IL-8 Production in Gastric Cancer Cells MKN28 cells were cultured in the complete medium in 24-well plates. After the cells reached subconuency, teprenone (CAS 6809-52-5; compliments of Eisai Ltd, Tokyo, Japan) was added at a nal concentration of 1, 10 or 50 mol/l. Two hours after addition of teprenone, H. pylori was added at a nal concentration of 1.0 107 colony-forming units (CFU)/ml, and then MKN28 cells were incubated at 37 C in 5% CO 2 for 24 hours. The cell supernatants were collected by centrifugation at 8,500 g for 3 minutes to remove bacteria and stored at 20 C until assays. IL-8 levels in the culture supernatant were determined by ELISA (R&D Systems, Minneapolis, MN). Statistical Analysis Signicant differences between continuous variables were determined using two-tailed paired Students t-test. Multiple comparisons were analyzed by one-way analysis of variance followed by Fishers projected least signicant difference (PLSD) test. Data analyses were performed with a standard biomedical software package (StatView J-4.5; Abacus Concepts, Inc., Berkeley, CA), and

Miyake et al. differences with a p-value less than .05 were regarded as statistically signicant.
Results

Effect of Teprenone, Sucralfate or H2-Receptor Blocker on H. pylori Gastritis and Serological Findings Baseline characteristics for all three groups are shown in Table 1. There were no signicant differences between groups in age, sex, smoking habits, alcohol intake, serum anti-H. pylori IgG titer, or gastrin and pepsinogen I and II levels. There were also no signicant differences between groups in baseline histological parameters. Signicant decreases in neutrophil inltration and H. pylori density in the corpus were noted in the teprenone group 3 months after the treatment (before vs. after, 2.49 0.22 vs. 2.15 0.23, p = .009; 2.36 0.25 vs. 2.00 0.24, p = .035, respectively). By contrast, in the sucralfate and H2RA groups, no change in histological parameters was seen 3 months after treatment (Table 2). Serological ndings showed a signicant decrease in pepsinogen I/II ratios in the teprenone group (before vs. after, 3.00 0.27 vs. 2.71 0.26, p = .022) but not in the sucralfate and H2-RA groups (Fig. 1). In Fig. 2a and b, we show post- to pretreatment

Table 1 Baseline characteristics of patients with H. pylori infection (clinico-serological parameters)

Parameter Mean age (SD) Sex (% male) HP IgG titer (SD) Pepsinogen I/II (SD) Pepsinogen I (SD) Pepsinogen II (SD) Gastrin (SD) Smoking (cigarettes/day) Alcohol (ml/day) Teprenone group 55.7 (2.1) 10 (43.5) 380.3 (63.4) 2.98 (0.32) 81.4 (18.7) 26.8 (4.7) 119.1 (24.3) 4.4 (2.1) 15.1 (6.0) Sucralfate group 58.5 (2.5) 10 (45.5) 342.1(84.4) 2.90 (0.32) 90.2 (21.3) 34.4 (10.6) 118.2 (38.0) 5.8 (2.3) 15.1 (6.1) H2 blocker group 56.4 (2.7) 12 (52.2) 421.0 (57.2) 2.85 (0.29) 70.8 (16.6) 25.6 (4.28) 114.8 (27.5) 3.4 (1.5) 22.5 (6.3) p-Value NS NS NS NS NS NS NS NS NS

Table 2 Score of histological parameters based on the updated Sydney system; comparison between values before and after treatment
Teprenone group Parameter Antral activity (SD) Corpus activity (SD) Antral inammation (SD) Corpus inammation (SD) Antral H. pylori (SD) Corpus H. pylori (SD)
ap

Sucralfate group Before 2.10 (0.26) 2.30 (0.16) 2.32 (0.17) 2.18 (0.12) 2.00 (0.23) 2.33 (0.16) After 2.11 (0.24) 2.48 (0.14) 2.15 (0.15) 2.23 (0.10) 2.18 (0.26) 2.43 (0.13)

H2-RA group Before 2.31 (0.20) 2.23 (0.12) 2.23 (0.15) 2.18 (0.09) 2.00 (0.21) 2.31 (0.16) After 2.18 (0.22) 2.36 (0.12) 2.09 (0.16) 2.20 (0.10) 1.81 (0.22) 2.36 (0.16)

Before 2.37 (0.24) 2.49 (0.22) 2.37 (0.17) 2.33 (0.17) 2.16 (0.25) 2.36 (0.25)

After 2.36 (0.25) 2.15 (0.23)a 2.34 (0.19) 2.11 (0.14) 2.05 (0.28) 2.00 (0.24)b

= .009; bp = .035.

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Effects of Teprenone on H. pylori Gastritis

133

Figure 1 The effects of teprenone, sucralfate or H2-receptor blocker on the serum pepsinogen I/ II ratio. Serum pepsinogen I and II levels were measured by a specic RIA. Sera were collected prior to the start of treatment (pretreatment) and 3 months after treatment (posttreatment).

Figure 3 The effects of teprenone, sucralfate or H2-receptor blocker on gastrin level. Serum gastrin levels were measured by a specic RIA. Sera were collected prior to the start of treatment (pretreatment) and 3 months after treatment (posttreatment).

ratios for pepsinogen I and II levels in each group. Although there were no signicant differences in the ratios for either pepsinogen I or pepsinogen II in each group, both pepsinogens tended to decrease in the teprenone group, as compared to the H2-RA group which showed a tendency to increase for both pepsinogens. Figure 3 shows changes between pre- and posttreatment levels of gastrin in each group. In

the H2-RA group, gastrin had a tendency to increase, although not signicantly. On the other hand, no change was observed in either the teprenone or sucralfate group. There were no signicant differences in serum anti-H. pylori IgG titer between pre- and posttreatment levels for each group (data not shown).

Figure 2 The effects of teprenone, sucralfate or H2-receptor blocker on serum pepsinogen I (a) or II (b) level, respectively, represented as the ratio of posttreatment level to pretreatment level. Serum pepsinogen I and II levels were measured by a specic RIA. Sera were collected prior to the start of treatment (pretreatment) and 3 months after treatment (posttreatment).
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134

Miyake et al.

Figure 4 Effects of teprenone on IL8 production induced in gastric epithelial cells (MKN28 cells) cocultured with H. pylori. MKN28 cells were incubated for 24 hours in the presence or absence of H. pylori or teprenone at nal concentrations of 1, 10, or 50 mol/l. IL-8 levels in the supernatants were determined by ELISA.

Effect of Teprenone on IL-8 Production by MKN28 Cells Cocultured with H. pylori Next, to clarify the mechanism by which teprenone reduces both neutrophil inltration and H. pylori density scores of the corpus, we examined the effect of teprenone on IL-8 production, a key factor for neutrophil accumulation in the gastric mucosa, in MKN28 gastric epithelial cells in vitro. We examined the production of IL-8 by MKN28 cells cultured with or without H. pylori. MKN28 cells cultured without H. pylori did not induce IL-8 production, but MKN28 cells cocultured with H. pylori signicantly induced IL-8 production. Teprenone inhibited H. pylori-enhanced IL-8 production in a dose-dependent manner. However, it did not affect basal IL-8 production in MKN28 cells cultured without H. pylori (Fig. 4).
Discussion

In the present study, we found for the rst time that teprenone reduced neutrophil inltration and H. pylori colonization in corpus gastritis mucosa infected with H. pylori, while H2-RA and sucralfate had no signicant effect. In addition, we showed that teprenone reduced H. pyloristimulated IL-8 release from gastric epithelial cells in vitro, a possible explanation for its effect on neutrophil inltration in vivo. We examined the effects of teprenone in H. pylori-associated gastritis, and compared the effects of teprenone with those of H2-RA and

sucralfate, following a 3-month treatment. In the present study, long-term teprenone treatment reduced neutrophil inltration and H. pylori colonization of the corpus. Teprenone is an acyclic polyisoprenoid compound. Its protective effect on the gastric mucosa has been shown in studies of gastric injury induced by cold- or water immersion-restraint stresses, and by NSAIDs [1722]. The cytoprotective action of teprenone is thought to occur through its induction of mucous secretion, suppression of lipid peroxide formation or inhibition of xanthine-xanthine oxidase; all independent of acid suppression [23,24]. It has also been shown that teprenone decreases myeloperoxidase activity, an indicator of neutrophil inltration in the gastric mucosa during restraint stress [25]. These ndings correspond well to the inhibitory effect of teprenone on neutrophil inltration in H. pylori-associated corpus gastritis in the present study. We could not explain exactly why corpus gastritis but not antral gastritis was improved by teprenone, as we could not explain exactly why most of the H. pylori gastritis pattern in Japan is corpus dominant, differing from that in Western countries. However, it is possible that action points of teprenone may be mainly in the fundic gland. Furthermore, another reason why teprenone seems to affect only the corpus may be the difference in the number of biopsies in the antrum and corpus. We estimated corpus gastritis using two biopsy sites (lesser and greater curvature) on the corpus. On the other hand, antral gastritis was estimated using one biopsy site (greater
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Effects of Teprenone on H. pylori Gastritis curvature) on the antrum. Actually, the inammation or activity score of the antrum showed a tendency to improve after teprenone treatment, although this was not statistically signicant. Further work is required before concluding that teprenone improves corpus gastritis but not antral gastritis. The antibacterial effect of teprenone on H. pylori has also been reported [26]. However, the minimum inhibitory concentration (MIC) of teprenone for H. pylori is relatively high, and so it is unlikely that the antibacterial action of teprenone directly affects H. pylori colonization density, at the doses used in the present study. We therefore speculated that the decrease of H. pylori colonization density produced by teprenone in the corpus might be accounted for by other mechanisms. Macrophages and epithelial cells in the gastric mucosa infected with H. pylori express IL-8, a key cytokine involved in neutrophil inltration in the stomach [6,27 ]. In fact, it has been shown that IL-8 levels correlate with H. pylori density and intensity of neutrophil inltration in the gastric mucosa [28]. Therefore, we evaluated the effect of teprenone on IL-8 production induced by H. pylori stimulated gastric epithelial cells in vitro. In the present study, teprenone exhibited dose-dependent inhibition of H. pylori-enhanced IL-8 production. The inhibition rate was up to approximately 50% for 0.05 mmol/l teprenone. Recently, Yoshimura et al. have also reported that teprenone at 0.1 mmol/l inhibits IL-8 mRNA expression in H. pylori-infected gastric mucosal cells (KATOIII cells) up to approximately 50% [29]. Based on our in vitro ndings for teprenone, it is likely that orally administered teprenone actually inhibits IL-8 production in the gastric mucosa, because it has been conrmed that therapeutic oral doses of teprenone will reach 0.010.1 mmol / l in the mucosa [30]. In contrast, H2-RA showed no signicant effect on H. pylori gastritis, although corpus gastritis scores tended to rise and antral gastritis scores to decline. In recent years, there have been several publications showing that long-term acid suppression therapy aggravates corpus gastritis, which might then progress into atrophic gastritis of the corpus [6]. Meining et al. have reported that lansoprazol treatment for 6 months leads to signicant aggravation of gastritis in the corpus, while partially improving antrum gastritis in patients with duodenal ulcers [31]. Other studies have also shown resolution of antral and aggra 2004 Blackwell Publishing Ltd, Helicobacter, 9, 130 137

135 vation of corpus gastritis after 8 weeks of omeprazole treatment [32,33]. All these studies suggest that proton pump inhibitor (PPI) treatments might change the distribution of H. pylori colonization as it changes pH within the stomach. In addition to PPIs, it has also been suggested that long-term treatment with H2-RA may have similar aggravating effects on neutrophil inltration in the corpus [31,34]. Our results, showing a tendency for a slight increase in corpus gastritis activity following a 3-month H2-RA treatment, are consistent with these previous results. Sucralfate is an effective, well-known cytoprotective agent for peptic ulcers, with effects comparable to those of H2-RA [35,36]. Banerjee et al. have reported that 4-week treatment with sucralfate suppresses antral H. pylori colonization and gastric urease activity in patients with duodenal ulcer [37]. On the other hand, in the present study we found that sucralfate had no effect on antral H. pylori colonization, including gastritis scores. However, in terms of the effect of sucralfate on H. pylori colonization, Banerjee et al. looked at duodenal ulcer patients with antral predominant type gastritis, whereas we mostly examined subjects with corpus predominant type gastritis. Uemura et al. have reported that changes in the grade of H. pylori colonization and neutrophil inltration were classied according to severity of corpus gastritis before treatment [1,28,38]. Thus, discrepancies in the effect of sucralfate between the Banerjee et al. study and our study might be due to the different types of gastritis our respective studies focused on. In the present study, not only did we nd that pepsinogen I and II showed a tendency to decrease in the teprenone group, but we also found a signicant decrease in their ratio. Successful eradication of H. pylori infection has been shown to result in a signicant fall in serum pepsinogen II and a concomitant improvement of histological gastritis [3941]. On the other hand, changes in serum pepsinogen I levels after eradication are controversial, although most studies have shown decreases in pepsinogen I levels as well [3942]. Kreuning et al. have shown that serum pepsinogen I correlates with activity of corpus gastritis [43]. Therefore, the tendency for both pepsinogen I and II to decrease in the teprenone group might be a result of the predominant improvement of corpus gastritis by teprenone treatment. These results seem to support the aforesaid effects of teprenone on histological corpus gastritis.

136 In conclusion, long-term teprenone treatment in patients with H. pylori infection results in decreases of neutrophil inltration and H. pylori density in the corpus, but not in the antrum. Corpus-dominant gastritis often leads to multifocal atrophy and intestinal metaplasia thought to be associated with an increased risk of gastric cancer. Especially, hypochlorhydria induced by corpus atrophy is related to carcinogenesis. Japan has a high rate of H. pylori infection. It should be stressed that the best treatment for H. pylori gastritis is eradication of H. pylori infection. However, because the risk of gastric cancer is limited to 5 percent at 10 years, as Uemura N et al. showed by Kaplan-Meier analysis, [44] not all H. pylori-infected patients have to be eradicated for prevention of gastric cancer. We may require picking up high risk patients for gastric cancer. At least, in the case that eradication is not necessary, or not possible, long-term teprenone treatment would be useful. Further work is clearly required before the conclusion can be reached that long-term treatment with teprenone may have benecial effects on outcomes such as gastric cancer occurrence in corpuspredominant gastritis.
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