Introduction

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The compound N-(3-aminopropionyl)-L-histidinato zinc (polaprezinc), a chelate of zinc and L-carnosine, is an antiulcer agent developed in Japan (Fig. 1) (Ueki et al., 1989). It is known that carnosine increases granulation tissue (Vizioli and Almeida, 1978; Nagai et al., 1986) and accelerates gastric ulcer healing in rats (Yamakawa, 1975). Zinc has been reported to have protective action against various experimental gastric lesions (Cho et al., 1976; Cho and Ogle, 1977, 1978), and clinical studies have shown antiulcer action of zinc in humans (Frommer, 1985; Alcala-Santaella et al., 1985; Varas-Lorenzo, 1986). Polaprezinc was originally designed to combine the beneficial effects of zinc and carnosine. Several reports have shown protective action of polaprezinc against experimental gastric lesions induced by various noxious agents (Ueki et al., 1989; Arakawa et al., 1990; Cho et al., 1991; Yoshikawa et al., 1991b; Hiraishi et al., 1999) and that polaprezinc accelerates gastric ulcer healing in humans (Morise et al., 1992). Although the mechanisms of antiulcer action of polaprezinc could be partly explained by its stimulative effect on mucus secretion, membrane-stabilizing effect, and antioxidant properties (Arakawa et al., 1990; Cho et al., 1991; Yoshikawa et al., 1991a), they are not fully understood.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Chemical structure of N-(3-aminopropionyl)-L-histidinato zinc (polaprezinc).

A growing amount of evidence shows that epithelial cells are playing an important role in the gastric mucosal cytokine network (Crabtree, 1996; Shimada and Terano, 1998). In Helicobacter pylori infection, gastric epithelial cells secrete large amounts of interleukin (IL)-8, a prototype of the CXC chemokines that recruit and activate neutrophils (Harada et al., 1996), in response to proinflammatory cytokines produced locally or in response to attachment of H. pylori to the cell surface (Crabtree et al., 1994). This epithelial chemokine response appears to be a primary factor in the induction of gastric inflammation in response to H. pylori infection. IL-8 mRNA expression is regulated by the transcription factor nuclear factor-B (NF-B) in combination with NF-IL-6 or activating protein-1 (AP-1) (Mukaida et al., 1990; Yasumoto et al., 1992; Matsusaka et al., 1993; Aihara et al., 1997). It has been shown that inflammatory cytokines, such as tumor necrosis factor  (TNF-) and IL-, and H. pylori attachment to the cell surface activate NF-B in gastric epithelial cells (Aihara et al., 1997; Keates et al., 1997). Because sustained inflammation is associated with mucosal damage and perhaps gastric carcinogenesis, agents that down-regulate NF-B activity and IL-8 expression may be of therapeutic significance. In this study, we examined whether polaprezinc affects NF-B activity and IL-8 expression in NKN28 gastric epithelial cells.

	Materials and Methods

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Reagents. Polaprezinc was a gift from Zeria Pharmaceuticals (Tokyo, Japan). Recombinant human TNF- and human IL-1 were purchased from R&D Systems, Inc. (Minneapolis, MN). ZnSO₄, L-carnosine, H₂O₂, and WST-1 assay reagent were obtained from Wako Pure Chemical (Osaka, Japan). Phorbol-12-myristate-13 acetate (PMA) was obtained from Research Biochemicals Inc. (Natick, MA).

Cell Culture. MKN28, a cell line derived from moderately differentiated gastric carcinoma (Motoyama et al., 1986) was obtained from Health Science Research Resources Bank (Osaka, Japan) and used as a model of gastric epithelial cells. Cells were routinely grown in Ham's F-12 culture medium (Gibco Laboratories, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Gibco Laboratories). Fetal bovine serum was eliminated 24 h before initiating experiments.

Quantitation of Secreted IL-8. MKN28 cells were cultured in 24-well dishes (2 × 10⁵ cells/well). Cells were incubated with TNF- or IL-1 in the absence and in the presence of polaprezinc for 16 h before collecting the culture supernatant of each well. The concentration of IL-8 in the culture supernatant (0.5 ml) was determined by human IL-8 specific enzyme-linked immunosorbent assay kits (BioSource Int'l, Camarillo, TX).

Northern Blot Analysis. After incubation with test agents, MKN28 cell monolayers grown in 10-cm dishes were scraped, and total RNA was extracted with Trizol reagent (Gibco Laboratories). Total RNA (20 µg/lane) was separated on a 1% agarose denaturing gel containing 0.66 M formaldehyde (Formalin) and transferred to Hybond-N⁺ nylon filters (Amersham Pharmacia Biotech, Uppsala, Sweden). An IL-8 cDNA probe was made from reverse transcribed polymerase reaction product (PCR) with mRNA extracted from TNF--stimulated MKN28 cells. The primers used were as follows: 5'-ATGACTTCCAAGCTGGCCGTGGCT-3' (forward primer) and 5'-CTCAGCCCTCTTCAAAAACTTCTC-3' (reverse primer) (PCR product 291 bp). PCR products were purified with Qiaquick PCR purification kit (Quiagen, Hilden, Germany), and sequence identity was confirmed by direct sequencing. Hybridization with human glyceraldehyde-3-phosphate dehydrogenase cDNA probe (Clonetech, Palo Alto, CA) also was performed for standardization.

Electrophoretic Mobility Shift Assay (EMSA). Nuclear extracts were prepared from ~5 × 10⁶ cells as described by Schreiber et al. (1989) with some modifications. After incubation with test agents, MKN28 cells were harvested and washed twice with ice-cold PBS. Cells were lysed in 800 µl of hypotonic buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 2 mM MgCl₂, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride] on ice for 15 min, after which 50 µl of 10% Nonidet P-40 solution was added, and the mixture was vortexed vigorously for 15 s and centrifuged for 30 s at 12,000 rpm. The pelleted nuclei were resuspended in 50 µl of buffer B [50 mM HEPES (pH 7.9), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, and 10% (v/v) glycerol] and incubated on ice for 20 min with intermittent mixing. The tubes were centrifuged for 5 min at 12,000 rpm, and the supernatant containing nuclear extracts was collected. The protein concentration was determined with a Bio-Rad protein concentration assay kit (Bio-Rad Laboratories, Richmond, CA). The nuclear extracts were stored at 70°C until use.

Western Blot Analysis. MKN28 cells grown in 10-cm culture dishes were treated with test agents and directly collected in 150 µl of SDS-sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 25% glycerol, 5% -mercaptoethanol, and 0.01% bromophenol blue. The cell lysates were sonicated for 15 s to shear high-molecular-weight DNA and reduce sample viscosity. After 10% SDS-polyacrylamide gel electrophoresis, the separated proteins were transferred onto a polyvinylidene difluoride membrane (Clear blot membrane-P; ATTO, Tokyo, Japan). Polyvinylidene difluoride membranes were blocked in Tris-buffered saline/Tween 20 (TBST) [20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% Tween 20] containing 5% nonfat dry milk (Bio-Rad Laboratories) overnight at 4°C. The membranes were then incubated with rabbit polyclonal antiphospho-IB- (Ser32) antibody (New England BioLabs, Beverly, MA) or rabbit polyclonal anti-IB- antibody (New England BioLabs) in TBST containing 5% BSA overnight at 4°C. After washing with TBST three times for 5 min, the membranes were incubated with peroxidase-conjugated anti-rabbit IgG antibody (New England BioLabs) in TBST containing 5% nonfat dry milk for 1 h at room temperature. The membranes were washed again, and the specific bands were visualized with LumiGLO chemiluminescent reagent (New England BioLabs).

Statistics. Data were expressed as the mean ± S.D. Statistical analysis was performed using Student's t test for unpaired data, and p values <.05 were considered to be significant.

	Results

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Effect of Polaprezinc on Proinflammatory Cytokine-Induced IL-8 Secretion. At first, we examined the effect of polaprezinc on IL-8 secretion induced by TNF- or IL-1 in MKN28 cells. In the experiments shown in Fig. 2A, cells were serum-deprived for 24 h and treated with 10 ng/ml TNF- alone or with increasing concentrations of polaprezinc (10-1000 µM). Each concentration of polaprezinc was preincubated with the cells for 3 h before the addition of TNF-, and TNF- treatment was performed in the constant presence of polaprezinc. Cell-free supernatants were harvested at a 16-h time point, and the IL-8 concentration of each sample was measured with human IL-8 specific enzyme-linked immunosorbent assay. Incubation with 10 ng/ml TNF- caused a significant increase in IL-8 secretion by MKN28 cells (an ~14-fold increase). However, compared with the IL-8 levels measured in the presence of 10 ng/ml TNF- alone, polaprezinc caused a dose-dependent reduction in IL-8 secretion by MKN28 cells. A significant suppression in the IL-8 levels was observed in the presence of as low as 10 µM polaprezinc. In this series of experiments, 10, 100, 300, and 1000 µM polaprezinc caused suppression of the IL-8 secretion induced by 10 ng/ml TNF- by ~57, 60, 96, and 96%, respectively.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Effect of polaprezinc on TNF--induced IL-8 secretion in MKN28 cells. A, polaprezinc suppressed TNF- (10 ng/ml)-induced IL-8 secretion in a dose-dependent manner. Control, medium alone; T10, TNF- (10 ng/ml); T10/P10, TNF- (10 ng/ml) + 10 µM polaprezinc; T10/P100, TNF- (10 ng/ml) + 100 µM polaprezinc; T10/P300, TNF- (10 ng/ml) + 300 µM polaprezinc; T10/P1000, TNF- (10 ng/ml) + 1000 µM polaprezinc (n = 4-6). *p < .01 versus TNF-. B, effect of 300 µM polaprezinc on IL-8 secretion induced by various concentrations (0.1-100 ng/ml) of TNF-. Control, medium alone control; T100, 100 ng/ml TNF-; T10, 10 ng/ml TNF-; T1, 1 ng/ml TNF-; T0.1, 0.1 ng/ml TNF- (n = 4-6). *p < .01 versus polaprezinc ().

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Effect of polaprezinc on IL-1-induced IL-8 secretion in MKN28 cells. A, polaprezinc suppressed IL-1 (10 ng/ml)-induced IL-8 secretion in a dose-dependent manner. Control, medium alone; B10, IL-1 (10 ng/ml); B10/P10, IL-1 (10 ng/ml) + 10 µM polaprezinc; B10/P100, IL-1 (10 ng/ml) + 100 µM polaprezinc; B10/P300, IL-1 (10 ng/ml) + 300 µM polaprezinc; B10/P1000, IL-1 (10 ng/ml) + 1000 µM polaprezinc (n = 4-6). *p < .01 versus IL-1. B, effect of 300 µM polaprezinc on IL-8 secretion induced by various concentrations (0.1-100 ng/ml) of IL-1. Control, medium alone; B100, 100 ng/ml IL-1; B10, 10 ng/ml IL-1; B1, 1 ng/ml IL-1, B0.1, 0.1 ng/ml IL-1 (n = 4-6). *p < .01 versus polaprezinc ().

Effect of Polaprezinc on Proinflammatory Cytokine-Induced IL-8 mRNA Expression. Northern blot analysis was performed to determine whether polaprezinc down-regulates TNF-- or IL-1-induced IL-8 mRNA expression in MKN28 cells. Cells were incubated with 10 ng/ml TNF- or IL-1 in the presence and in the absence of increasing concentrations of polaprezinc. Polaprezinc was preincubated for 3 h before the addition of TNF- or IL-1, and the treatment of these proinflammatory cytokines was performed in the presence of polaprezinc. At a 4-h time point, culture supernatants were aspirated, and the cell monolayers were scraped. Total RNA was extracted, and standard Northern blot experiments were performed.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   A, Northern blot analysis showing effect of polaprezinc on TNF- (10 ng/ml)-induced IL-8 mRNA expression in MKN28 cells. Lane 1, medium alone; lane 2, TNF- (10 ng/ml); lane 3, TNF- (10 ng/ml) + 10 µM polaprezinc; lane 4, TNF- (10 ng/ml) + 100 µM polaprezinc; lane 5, TNF- (10 ng/ml) + 300 µM polaprezinc; lane 6, TNF- (10 ng/ml) + 1000 µM polaprezinc. B, densitometric analysis of the experiment shown in Fig. 4A.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   A, Northern blot analysis showing effect of polaprezinc on IL-1 (10 ng/ml)-induced IL-8 mRNA expression in MKN28 cells. Lane 1, medium alone; lane 2, IL-1 (10 ng/ml); lane 3, IL-1 (10 ng/ml) + 10 µM polaprezinc; lane 4, IL-1 (10 ng/ml) + 100 µM polaprezinc; lane 5, IL-1 (10 ng/ml) + 300 µM polaprezinc; lane 6, IL-1 (10 ng/ml) + 1000 µM polaprezinc. B, densitometric analysis of the experiment shown in Fig. 5A.

Effect of Polaprezinc on Proinflammatory Cytokine-Induced IL-8 NF-B Activation. Because IL-8 expression is mainly regulated by the transcription factor NF-B (Mukaida et al., 1990; Yasumoto et al., 1992; Matsusaka et al., 1993; Aihara et al., 1997), EMSA experiments were performed to examine the effect of polaprezinc on the activation of NF-B induced by proinflammatory cytokines. Incubation of MKN28 cells with TNF- (10 ng/ml) for 90 min caused significant up-regulation of NF-B-specific DNA-binding activity (Fig. 6). The retarded band was shifted to higher molecular weight when the nuclear extracts were preincubated with antibodies to the NF-B subunits p50 or p65, whereas preincubation with antibodies to the NF-B subunits p52 or c-Rel had no effect, indicating that NF-B in MKN28 cells mainly exists as a heterodimer of p50 and p65. The results of the supershift assay are confirmation that the observed bands were indeed NF-B. The disappearance of the retarded band in the presence of a 100-fold excess amount of unlabeled NF-B consensus oligonucleotide reconfirms that the band was NF-B.

View larger version (137K): [in this window] [in a new window]   Fig. 6.   EMSA showing effects of TNF- (10 ng/ml) and antibodies to the NF-B subunits on NF-B-specific DNA-binding activity. Lane 1, nuclear extract (); lane 2, medium alone; lane 3; TNF- (10 ng/ml); lane 4, TNF- (10 ng/ml) + anti-p50 antibody; lane 5, TNF- (10 ng/ml) + anti-p52 antibody; lane 6, TNF- (10 ng/ml) + anti-p65 antibody; lane 7, TNF- (10 ng/ml) + anti-c-Rel antibody; lane 8, TNF- (10 ng/ml) + 100-fold amount of cold competitor oligo: lane 9, TNF- (10 ng/ml) + a 100-fold amount of cold noncompetitor oligo.

View larger version (110K): [in this window] [in a new window]   Fig. 7.   EMSA showing effect of polaprezinc on NF-B activation induced by TNF- (10 ng/ml). Lane 1, nuclear extract (); lane 2, medium alone; lane 3, TNF- (10 ng/ml); lane 4, TNF- (10 ng/ml) + 10 µM polaprezinc; lane 5, TNF- (10 ng/ml) + 100 µM polaprezinc; lane 6, TNF- (10 ng/ml) + 300 µM polaprezinc; lane 7, TNF- (10 ng/ml) + 1000 µM polaprezinc; lane 8, TNF- (10 ng/ml) + a 100-fold amount of cold competitor oligo; lane 9, TNF- (10 ng/ml) + 100-fold amount of cold noncompetitor oligo.

View larger version (85K): [in this window] [in a new window]   Fig. 8.   EMSA showing effect of polaprezinc on the NF-B activation induced by PMA or H2O2 in MKN28 cells. Lane 1, medium alone; lane 2, 0.1 µM PMA; lane 3, 0.1 µM PMA + 300 µM polaprezinc; lane 4, 0.1 µM PMA + a 100-fold amount of cold competitor oligo; lane 5, 1 mM H2O2; lane 6, 1 mM H2O2 + 300 µM polaprezinc; lane 7, 1 mM H2O2 + a 100-fold amount of cold competitor oligo.

View larger version (83K): [in this window] [in a new window]   Fig. 9.   EMSA showing effects of ZnSO4 and L-carnosine on TNF- (10 ng/ml)-induced activation of NF-B in MKN28 cells. Lane 1, medium alone; lane 2, TNF- (10 ng/ml); lane 3, TNF- (10 ng/ml) + 100 µM ZnSO4; lane 4, TNF- (10 ng/ml) + 300 µM ZnSO4; lane 5, TNF- (10 ng/ml) + 100 µM L-carnosine; lane 6, TNF- (10 ng/ml) + 300 µM L-carnosine; lane 7, TNF- (10 ng/ml) + a 100-fold amount of cold competitor oligo.

Effect of Polaprezinc on IB- Phosphorylation Induced by TNF-. In the cytosol, NF-B is inactive when complexed with IB proteins (Baeuerle and Baltimore, 1996). Phosphorylation of IB- at Ser32 and 36 triggers dissociation of IB from NF-B complex, which results in NF-B activation and translocation into the cell nucleus (Brown et al., 1995; Traenckner et al., 1995). Dissociated IB proteins are subjected to proteasome-mediated degradation (Brown et al., 1995; Traenckner et al., 1995). We examined the effect of polaprezinc on TNF- (10 ng/ml)-induced IB- phosphorylation (Ser32) in MKN28 cells. Cells were preincubated with 300 µM polaprezinc for 3 h and then treated with TNF- (10 ng/ml) for 5, 10, 15, and 30 min in the constant presence of 300 µM polaprezinc. Control cells were similarly treated with TNF- in the absence of polaprezinc. Western blot analysis was performed with anti-phospho-IB- (Ser32) antibody and anti-IB- antibody.

View larger version (30K): [in this window] [in a new window]   Fig. 10.   Western blot analysis of cell extracts from MKN28 cells treated with TNF- (10 ng/ml), with anti-phospho-IB- (Ser32) antibody and anti-IB- antibody in the absence (A) and in the presence (B) of polaprezinc (300 µM).

Effect of Polaprezinc on AP-1 Activity. Because AP-1 also may affect IL-8 expression (Yasumoto et al., 1992), we examined the effect of polaprezinc on AP-1-specific DNA-binding activity in MKN28 cells. As shown in Fig. 11, AP-1 activity was present in control cells, and it was slightly up-regulated by incubation with 10 ng/ml TNF-, 10 ng/ml IL-1, or 1 mM H₂O₂, whereas PMA had a more potent effect. Preincubation of the cells with 300 µM polaprezinc for 3 h completely abolished AP-1 activity, indicating that polaprezinc also down-regulates AP-1 activity in addition to NF-B.

View larger version (98K): [in this window] [in a new window]   Fig. 11.   EMSA showing effect of polaprezinc on AP-1 activity. Lane 1, medium alone; lane 2, 300 µM polaprezinc; lane 3, TNF- (10 ng/ml); lane 4, TNF- (10 ng/ml) + 300 µM polaprezinc; lane 5, IL-1 (10 ng/ml); lane 6, IL-1 (10 ng/ml) + 300 µM polaprezinc; lane 7, 0.1 µM PMA; lane 8, 0.1 µM PMA + 300 µM polaprezinc; lane 9, 1 mM H2O2; lane 10, 1 mM H2O2 + 300 µM polaprezinc; lane 11, medium alone + a 100-fold amount of competitor oligo; lane 12, 0.1 µM PMA + a 100-fold amount of competitor oligo.

	Discussion

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Polaprezinc is a chelate compound of zinc and L-carnosine (Ueki et al., 1989), and previous studies showed its usefulness as an antiulcer agent (Ueki et al., 1989; Arakawa et al., 1990; Cho et al., 1991; Yoshikawa et al., 1991b; Morise et al., 1992; Hiraishi et al., 1999). In an effort to understand molecular mechanisms of polaprezinc's action, we found in the present study that polaprezinc suppressed proinflammatory cytokine-induced IL-8 mRNA expression and secretion in MKN28 gastric epithelial cells. We also found that polaprezinc down-regulated NF-B activation induced by proinflammatory cytokines, phorbol ester, and H₂O_2. These results suggest that polaprezinc is a novel type of anti-inflammatory drug that can control gastric inflammatory responses.

The transcription factor NF-B regulates the expression of a wide range of genes involved in immune response, inflammation, and acute phase response, as well as several viral genes (Baeuerle and Henkel, 1994). Recent evidence also suggests that NF-B is involved in carcinogenesis and apoptosis (Sharma and Narayanan, 1996; Gilmore et al., 1996). NF-B is a multisubunit transcription factor and several proteins belong to the NF-B family. These include NF-B1 (p105/p50), NF-B2 (p100/p52), RelA (p65), RelB, and c-Rel proteins (Gilmore et al., 1996). NF-B proteins form homo- or heterodimers, and, most commonly, NF-B dimers are composed of p65 and p50 or p52. In MKN28 cells used in this study, NF-B dimers were mainly composed of p65 and p50 (Fig. 6). In the resting condition, NF-B dimers are present in the cytoplasm in an inactive form by binding to the inhibitory molecule IB. Upon stimulation, IB kinase or kinases phosphorylate IB, and this phosphorylation is followed by ubiquitination and degradation of IB (DiDonato et al., 1997). After dissociation of IB, free, activated NF-B complexes are translocated to the nucleus, and the binding of NF-B to the promoter region of target genes activates transcription. Because NF-B is involved in the expression of a wide range of genes relating the cellular inflammatory and immune responses, dysregulation of this transcription factor may be associated with various pathological conditions. Thus, NF-B is a potential target for the treatment of inflammatory diseases (Neurath et al., 1996; Papavassiliou, 1998).

Recent evidence shows that reactive oxygen intermediates are involved in NF-B activation, and binding of activated NF-B to its cognate DNA site also is reported to be redox (reduction-oxidation)-sensitive (Sen and Packer, 1996). Various kinds of antioxidants, such as N-acetylcysteine, dimethyl sulfoxide, and -lipoic acid, have been reported to down-regulate NF-B activation in a wide range of cell types (Sen and Packer, 1996). Our previous study also demonstrated that NF-B activation and IL-8 expression are redox-sensitive in MKN28 cells (Shimada et al., 1999). Because polaprezinc has been reported to have antioxidant properties (Yoshikawa et al., 1991b), the inhibitory effect of polaprezinc on NF-B activation and IL-8 expression observed in the present study may be explained by its antioxidant properties. Because polaprezinc suppressed IB- phosphorylation induced by TNF- treatment (Fig. 10), the primary site of polaprezinc's action should be IB kinase or upstream of IB kinase. If polaprezinc's action can be ascribed to its antioxidant properties, there may be redox-sensitive sites around IB kinase in the signaling pathway leading to NF-B activation. However, we cannot exclude the possibility that polaprezinc acts at multiple sites inside the cells.

We compared the effects of zinc and L-carnosine, components of polaprezinc, on NF-B activation (Fig. 9). ZnSO₄ (100-300 µM) had a potent inhibitory effect on TNF--induced NF-B activation, whereas L-carnosine (100-300 µM) had no significant effect. Thus, the observed inhibitory effect of polaprezinc on NF-B activation is likely to be mediated by its component, zinc. Zinc is known to have a variety of biological effects, including antioxidant properties (Yoshikawa et al., 1991a). Connell et al. (1997) showed that, in endothelial cells, zinc protects against cytokine-mediated activation of NF-B and AP-1, up-regulation of inflammatory cytokines, and endothelial dysfunction. Consistent with this study, we found that polaprezinc inhibits AP-1-specific DNA-binding activity in MKN28 cells (Fig. 11). AP-1 also is known to be involved in the expression of genes relating to inflammatory responses (Connell et al., 1997). It should be noted that AP-1 is another redox-sensitive transcription factor (Sen and Packer, 1996) and certain antioxidant compounds are capable of influencing AP-1 activity (Sen and Packer, 1996). Additional studies are needed to determine whether polaprezinc's inhibitory effect on AP-1 activity can be ascribed to its antioxidant properties. Yasumoto et al. (1992) reported that IL-8 expression is affected by AP-1 in addition to NF-B in certain cell types. Although we have not determined the relative importance of these transcription factors in terms of IL-8 expression in MKN28 cells, it is possible that the potent inhibitory effect of polaprezinc on IL-8 expression in this cell line is partly due to the inhibition of AP-1 activity.

When administered p.o., polaprezinc adheres to the surface of the gastric mucosal layers, and its adhesiveness to gastric mucosa is superior to that of ZnSO₄ or ZnSO₄ + L-carnosine (Seiki et al., 1992). Therefore, although NF-B is ubiquitously expressed in most tissues, polaprezinc may be useful as an agent to specifically down-regulate NF-B activation in the gastric mucosa and to normalize the dysregulation of gastric mucosal cells under prolonged inflammatory conditions, such as H. pylori-associated gastritis. This study may provide a theoretical basis for the use of this agent as a novel type of anti-inflammatory drug to control gastric inflammatory responses.