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Keywords:

  • Helicobacter pylori;
  • caffeic acid phenethyl ester;
  • chemoprevention;
  • gastritis;
  • Mongolian gerbils

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Nuclear factor-κB (NF-κB) plays a major role in host inflammatory responses and carcinogenesis and as such is an important drug target for adjuvant therapy. In this study, we examined the effect of caffeic acid phenethyl ester (CAPE), an NF-κB inhibitor, on Helicobacter pylori (H. pylori)-induced NF-κB activation in cell culture and chronic gastritis in Mongolian gerbils. In AGS gastric cancer cells, CAPE significantly inhibited H. pylori-stimulated NF-κB activation and mRNA expression of several inflammatory factors in a dose-dependent manner, and prevented degradation of IκB-α and phosphorylation of p65 subunit. To evaluate the effects of CAPE on H. pylori-induced gastritis, specific pathogen-free male, 6-week-old Mongolian gerbils were intragastrically inoculated with H. pylori, fed diets containing CAPE (0–0.1%) and sacrificed after 12 weeks. Infiltration of neutrophils and mononuclear cells and expression of NF-κB p50 subunit and phospho-IκB-α were significantly suppressed by 0.1% CAPE treatment in the antrum of H. pylori-infected gerbils. Labeling indices for 5′-bromo-2′-deoxyuridine both in the antrum and corpus and lengths of isolated pyloric glands were also markedly reduced at the highest dose, suggesting a preventive effect of CAPE on epithelial proliferation. Furthermore, in the pyloric mucosa, mRNA expression of inflammatory mediators including tumor necrosis factor-α, interferon-γ, interleukin (IL)-2, IL-6, KC (IL-8 homologue), and inducible nitric oxide synthase was significantly reduced. These results suggest that CAPE has inhibitory effects on H. pylori-induced gastritis in Mongolian gerbils through the suppression of NF-κB activation, and may thus have potential for prevention and therapy of H. pylori-associated gastric disorders. © 2009 UICC

Nuclear factor-κB (NF-κB) plays a central role in many physiological processes in the whole body such as immune responses, cell proliferation, and inflammation through promoting transcription of various cytokines, enzymes, chemokines, antiapoptotic factors and cell growth factors.1 Because many types of cancer, including neoplasm in the stomach, are known to be associated with chronic inflammation,2 inhibition of NF-κB activation has attracted increasing attention as a new therapeutic approach for chemoprevention of cancer development.3, 4 Several natural and synthetic compounds have been found to inhibit NF-κB activation, and to exert anti-inflammatory effects in vitro and in vivo.5, 6 Caffeic acid phenethyl ester (CAPE), one of the active components of propolis derived from honeybee hives, has been reported to be a selective inhibitor of NF-κB.7, 8 Besides that, recent study has also shown that CAPE may inhibit activator protein-1 (AP-1) activity in Helicobacter pylori (H. pylori)-stimulated gastric epithelial cells.9 Thus, further investigation was needed, to confirm how CAPE would influence many signal transduction cascades other than NF-κB pathway. Although the mechanisms of NF-κB inhibition by CAPE are not fully understood, research has demonstrated anti-inflammatory, anticarcinogenic and immunomodulatory effects of the compound in animal models.10–12

H. pylori is now recognized as a major causative factor for chronic gastritis and peptic ulcer, and there is compelling evidence indicating an association between H. pylori-induced chronic gastritis and development of stomach cancer.13, 14 Triple therapy with a proton pump inhibitor and 2 antimicrobials, amoxicillin and clarithromycin, is usually recommended as the general therapy for H. pylori eradication.15 However, considering the occurrence of antibiotic-resistance, the search for new agents for alternative therapies continues to be very important.16H. pylori infection also leads to activation of NF-κB signaling in gastric epithelial cells, and NF-κB-mediated cytokine expression is essentially involved with H. pylori-induced gastritis.17–21 Thus the degree of gastritis induced by a mutant strain of H. pylori lacking capacity for NF-κB activation was found to be lower than that with wild type infection.22 Inhibition of NF-κB could be a promising target for prevention and adjuvant therapy of H. pylori-associated gastric disorders.3, 23

The Mongolian gerbil (Meriones unguiculatus) provides a useful animal model of H. pylori-induced chronic active gastritis, allowing investigation of morbidity-related epithelial alterations in the gastric mucosa and their development into gastric neoplasia.24 We have previously demonstrated that some natural products in food such as a fruit-juice concentrate of Japanese apricot and nordihydroguaiaretic acid, an antioxidant to preserve food and oils, and canolol, a potent oxygen radicals scavenger contained in canola oil, have suppressive effects on H. pylori-induced gastric disorders in Mongolian gerbils.25–27 The purpose of this study was to evaluate possible anti-inflammatory effects of CAPE, a naturally-occurring compound in food, in the same model.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Chemicals and cell culture

CAPE was purchased from Cayman Chemicals (Ann Arbor, MI) (Fig. 1). AGS cells, the human gastric cancer cell line, were maintained in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (FCS, Sigma Chemical, St. Louis, MO), penicillin (100 units/ml), streptomycin (100 μg/ml) and amphotericin B (0.25 μg/ml) (Invitrogen, Carlsbad, CA). Culture dishes and plates were kept in an incubator with a humidified atmosphere of 5% CO2 at 37°C. CAPE was prepared as a 20 mg/ml solution in dimethyl sulfoxide immediately before use.

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Figure 1. Chemical structure of caffeic acid phenethyl ester. C17H16O4, molecular weight 284.3.

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Bacterial culture

H. pylori was prepared by the same method as described previously.28 Briefly, H. pylori strain ATCC43504 (American Type Culture Collection, Rockville, MD) was grown in Brucella broth (Becton Dickinson, Cockeysville, MD) containing 7% FCS, at 37°C under microaerobic conditions using an Anaero Pack Campylo (Mitsubishi Gas Chemical, Tokyo, Japan), at high humidity for 24 hr. The broth cultures of H. pylori were checked under a phase contrast microscope for bacterial shape and motility.

Luciferase reporter assay on transcriptional activation of NF-κB

To assess whether NF-κB is activated by H. pylori infection in gastric cancer cells and to determine the effects of CAPE, luciferase reporter assays were performed. AGS cells were cotransfected in a 24-well culture plate with two expression plasmids, one including a luciferase reporter gene under transcriptional control of the NF-κB element (pNF-κB-Luc; Stratagene, La Jolla, CA) and the other a transfection efficiency indicator (pGL4.74[hRluc/TK] Vector; Promega, Madison, WI) using the Lipofectamin 2000 (Invitrogen) transfection reagent. After 24 hr incubation, cells were challenged by infection with 1 × 106 colony-forming units (CFU)/well of H. pylori and immediately treated with various concentrations of CAPE (0, 10, 20, or 40 μg/ml) for 24 hr. NF-κB luciferase reporter gene assays were performed with a Dual Luciferase Reporter Assay System (Promega) and a luminometer (Lumat LB9501; Berthold, Bad Wildbad, Germany) according to the manufacturer's instructions.

Analysis of mRNA expression for inflammatory factors by relative quantitative real-time RT-PCR

To investigate the effects of CAPE on cytokine expression of H. pylori-stimulated AGS cells, real-time RT-PCR analysis was performed. AGS cells were challenged by infection with 1 × 107 colony-forming units (CFU)/dish of H. pylori and immediately treated with CAPE (0, 5, 10 or 20 μg/ml). After 24 hr incubation, total RNA was extracted from these cells using an RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). After DNase treatment, first strand cDNAs were synthesized using a SuperScript III First-Strand Synthesis System (Invitrogen) according to the manufacturer's instructions. Relative quantitative PCR of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-8, IL-10, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was carried out using a LightCycler system (Roche Diagnostics, Mannheim, Germany) with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as an internal control. The PCR was performed basically as described earlier using a QuantiTect SYBR Green PCR Kit (Qiagen).29 The primer sequences for each marker are listed in Table I. Specificity of the PCR reaction was confirmed using the melting program provided with the LightCycler software. To further confirm that there was no obvious primer dimer formation or amplification of any extra bands, the samples were electrophoresed in 3% agarose gels and visualized with ethidium bromide after the LightCycler reaction. Relative quantification was performed as previously established using the internal control without the necessity for external standards.29

Table I. Primer Sequences for Relative Quantitative Real-Time RT-PCR
SpeciesGeneSequencesProduct length (bp)Accession no.
  1. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TNF, tumor necrosis factor; IFN, interferon; IL, interleukin; iNOS, inducible nitric oxide synthase; COX, cyclooxygenase.

GerbilGAPDH5′-AACGGCACAGTCAAGGCTGAGAACG-3′118AB040445
5′-CAACATACTCGGCACCGGCATCG-3′
TNF-α5′-GCCCCCACCTCGTGCTCCTCAC-3′96AB177841
5′-GGCAGGGGCTCTTGATGGCAGACAG-3′
IFN-γ5′-AGAGCATAAACGCCATCAGG-3′120L37782
5′-TGCTCTGGATCTGTGGATCA-3′
IL-25′-AGCTCCTGAGAGGGATCAAC-3′139X68779
5′-ACATCATGCAGAGGTCCAAG-3′
IL-65′-ATGGCTGAAGTCCAAGACC-3′125AB164706
5′-GGAATGTCCTCAGCTTGGTA-3′
IL-105′-CAGGGCTCCTGAAAGAGTTA-3′114L37781
5′-AGAATGAGGTCAGGGGAATC-3′
iNOS5′-GCTTGAGCGAGGAGCAGGTTGAGGA-3′111AB177843
5′-CGCTGGCCTTTTTCACCCCATAGGA-3′
KC5′-CACCCGCTCGCTTCTTC-3′138AJ877921
5′-ATGCTCTTGGGGTGAATCC-3′
HumanGAPDH5′-GGGAAGCTTGTCATCAATGG-3′103NM_002046
5′-TGGACTCCACGACGTACTCA-3′
TNF-α5′-AGCCCATGTTGTAGCAAACC-3′135AF043342
5′-ATGAGGTACAGGCCCTCTGA-3′
IL-1β5′-AGGGACAGGATATGGAGCAA-3′127NM_000576
5′-TTCAACACGCAGGACAGGTA-3′
IL-85′-CGGAAGGAACCATCTCACTG-3′116NM_000584
5′-AGCACTCCTTGGCAAAACTG-3′
IL-105′-CCAAGACCCAGACATCAAGG-3′115NM_000572
5′-GGCCTTGCTCTTGTTTTCAC-3′
iNOS5′-CCCAAGCTCTACACCTCCAA-3′132AB022318
5′-TTTGAGCCTCATGGTGAACA-3′
COX-25′-CGCTTTATGCTGAAGCCCTA-3′127M90100
5′-TTTCTACCAGAAGGGCAGGA-3′

Western blot analysis

Total protein extract was obtained from H. pylori-stimulated and CAPE-treated AGS cells by a Nuclear Extract Kit (Active Motif, Carlsbad, CA). Protein samples were fractionated by SDS-PAGE and electrophoretically transferred to a PVDF membrane. Blots were blocked with 5% nonfat dry milk in tris-buffered saline for 1 hr and then incubated over night with a rabbit polyclonal anti-IκB-α antibody (Cell Signaling Technology, Beverly, MA), a mouse monoclonal anti-α-tubulin antibody (clone DM1A, Santa Cruz Biotechnology, Santa Cruz, CA), a rabbit polyclonal antiphospho-NF-κB p65 antibody (Ser276, Cell Signaling Technology) and a mouse monoclonal anti-actin antibody (clone ACTN05, Thermo Scientific, Fremont, CA). Detection was performed using an Immun-Star HRP Chemiluminescent Kit (Bio-Rad Laboratories, Hercules, CA).

In vivo experimental design

The experimental design is illustrated in Figure 2. A total of 55 specific pathogen-free male, 6-week-old Mongolian gerbils (Meriones unguiculatus; MGS/Sea, Kyudo, Fukuoka, Japan) were used. They were housed in plastic cages on hardwood-chip bedding in an air-conditioned biohazard room with a 12-hr light/12-hr dark cycle, and allowed free access to food and water throughout. The gerbils were divided into 6 groups (Groups A–F). Animals of Groups A–D were inoculated with 1.0 ml of broth culture containing H. pylori (1 × 108 CFU/ml) intragastrically using an oral catheter, while gerbils of Groups E and F were inoculated with Brucella broth alone. From weeks 2 to 12, the gerbils received CE-2 diets (CLEA Japan, Tokyo, Japan) containing CAPE at the concentrations of 0.1% (Groups A and E), 0.03% (Group B), 0.01% (Group C) and 0% (Groups D and F). All experimental diets were prepared at 8 day intervals in our laboratory and stored in a refrigerator. Food cups were replenished with fresh diet every second day. At week 12, all gerbils were intraperitoneally injected with 5′-bromo-2′-deoxyuridine (BrdU) at a dose of 100 mg/kg, 1 hr before sacrifice. The animals were then subjected to deep anesthesia and laparotomy with excision of the stomach, liver, spleen, kidney, heart and lung, and blood samples were collected from the inferior vena cava. The experimental design was approved by the Animal Care Committee of the Aichi Cancer Center Research Institute, and the animals were cared for in accordance with institutional guidelines as well as the Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan, June 1, 2006).

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Figure 2. Experimental design. Six-week-old male Mongolian gerbils were inoculated with Helicobacter pylori (ATCC43504 strain) or Brucella broth. After 2 weeks, animals were given basal diets (CE-2) containing caffeic acid phenethyl ester (CAPE) at various concentrations (0, 0.01, 0.03 and 0.1%) for 10 weeks.

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Histopathology and immunohistochemistry

The excised stomachs were fixed in 10% neutral-buffered formalin for 24 hr and sliced along the longitudinal axis into 4–8 strips of equal width, and embedded in paraffin. Serial paraffin sections were prepared and stained with hematoxylin and eosin (H&E) for morphological observation. The glandular mucosa of the antrum and corpus was examined histologically for inflammation and epithelial changes. The degree of chronic active gastritis was graded according to criteria modified from the Updated Sydney System,30 by scoring the infiltration of neutrophils and mononuclear cells, intestinal metaplasia and heterotopic proliferative glands, on a four-point scale (0–3; 0, normal; 1, mild; 2, moderate; 3, marked). Epithelial cell proliferation was assessed by BrdU labeling, visualized by immunostaining with a mouse monoclonal anti-BrdU antibody (clone Bu20a, diluted 1:1000, Dako, Glostrup, Denmark) as described previously.31 Labeling indices in BrdU-stained slides were determined as the mean percentages of BrdU-positive epithelial cells among total cells in 10 different randomly selected glands in both the antrum and corpus. Immunohistochemical analyses were carried out with a mouse monoclonal anti-COX-2 antibody (clone 33, diluted 1:100, BD Biosciences, San Jose, CA), a mouse monoclonal anti-phospho-IκB-α antibody (clone 5A5, diluted 1:150, Cell Signaling Technology) and a rabbit polyclonal anti-NFκB p50 antibody (clone H-119, diluted 1:100, Santa Cruz Biotechnology) as previously described.32–34 To quantitate the degree of staining, a grading system was employed with the following criteria: grade 0 (negative), grades 1–3 (increasing degrees of intermediate immunoreactivity) and grade 4 (extensive reactivity).

Gland isolation

Gland isolation was performed as previously described.35 Briefly, remaining portions of resected gastric mucosa were injected with calcium- and magnesium-free Hanks' balanced salt solution (HBSS) containing 30 mM ethylenediaminetetraacetic acid (EDTA) submucosally, incubated in EDTA-HBSS, and shaken for 15 min at 37°C. Then the mucosa was scraped off with a scalpel. Isolated glands were washed in phosphate buffered saline, fixed in 70% ethanol for a few hours, dehydrated with 95% ethanol, and stored at −20°C until use.

Analysis of mRNA expression in the pyloric mucosa of Mongolian gerbils

Relative quantitative real-time RT-PCR for TNF-α, interferon-γ (IFN-γ), IL-2, IL-6, IL-10, iNOS and IL-8 homologue (KC) was carried out using total RNA extracted from selected pyloric mucosal tissue with the gerbil-specific GAPDH gene as an internal control same as above. The expression levels of mRNAs were expressed relative to 1.0 in the control group (Group F).

Serology

Blood samples were centrifuged and separated sera were stored at −80°C until use. The titer of anti-H. pylori antibodies was measured using an ELISA kit (Biomerica, Newport Beach, CA) and values were expressed using an arbitrary index (AI).27 Sera were also used for measurement of gastrin levels (SRL, Tokyo, Japan).

Statistical analysis

Quantitative values were expressed as means ± SD or SE, and differences between means were statistically analyzed by ANOVA or Kruskal-Wallis followed by a multiple comparison test. p values of less than 0.05 were considered to be statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Suppressive effects of CAPE on H. pylori-induced NF-κB activation and mRNA expression of inflammatory factors in AGS cells

NF-κB activation in H. pylori-stimulated AGS cells was significantly increased as compared to that in noninfected control cells (p < 0.01) (Fig. 3). CAPE decreased the H. pylori-induced NF-κB transcriptional activation in a dose-dependent manner, with significance at the 20 and 40 μg/ml doses (p < 0.05 and p < 0.01, respectively). Relative quantitative real time RT-PCR data for mRNA expression of inflammatory cytokines and enzymes in the AGS cells are summarized in Figure 4. IL-8 mRNA expression in 20 μg/ml CAPE-treated cells was significantly suppressed as compared to H. pylori-stimulated control cells. Levels of TNF-α mRNA in 20 and 10 μg/ml CAPE-treated cells and IL-1β and iNOS mRNAs in all CAPE-treated cells were also markedly lower than in positive control. There were no significant differences in IL-10 and COX-2 expression among H. pylori-infected cells.

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Figure 3. Luciferase reporter assay for transcriptional activation of nuclear factor-κB (NF-κB) in the AGS gastric cancer cell line. Elevated transcriptional activation of NF-κB was induced after infection with Helicobacter pylori (H. pylori). Note the dose-dependent inhibition of NF-κB activation by caffeic acid phenethyl ester (CAPE) treatment compared with the vehicle control (0.1% dimethyl sulfoxide). Values are means ± SDs of data from three independent experiments.

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Figure 4. Relative expression levels of interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), IL-8 and IL-10 mRNAs in the AGS gastric cancer cell line. Values were set at 1.0 in untreated control and expressed as mean ± SE relative values. Note that the Y-axes have different scales. *p < 0.05 and **p < 0.01 vs. H. pylori-infected and CAPE-untreated samples.

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CAPE prevents IκB-α degradation and phosphorylation of NF-κB p65 in AGS cells

We assessed the degradation of IκB-α and phosphorylation of p65 subunit by Western blot analysis (Fig. 5). Western blotting showed that H. pylori stimulation up-regulated the phosphorylation of p65 and IκB-α degradation in AGS cells. CAPE treatment inhibited the phosphorylation of p65 and degradation of IκB-α in a dose-dependent manner.

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Figure 5. Western blotting for IκB-α and phospho-p65 in AGS cells. CAPE prevents IκB-α degradation and p65 phosphorylation in a dose-dependent manner. A representative data of three independent experiments is shown.

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Average body weights, relative organ weights and serological results

Data for average body weights, titers of anti-H. pylori antibodies, serum gastrin levels and relative organ weights are summarized in Table II. The average body weight in the 0.1% CAPE-treated and H. pylori-infected group (Group A) was significantly higher than for the other H. pylori-infected groups (Groups B–D) (p < 0.01). AI values for anti-H. pylori antibody titers and serum gastrin levels were markedly up-regulated by H. pylori infection (p < 0.01 and p < 0.05, respectively). There were no significant differences in the relative organ weights of liver and kidney between 0.1% CAPE-treated and noninfected gerbils (Group E) and untreated controls (Group F). The relative kidney weights in the H. pylori-infected group (Group D) were markedly higher than in Group F (p < 0.05). No macroscopic or microscopic lesions were observed in nonstomach internal organs, including the liver, spleen, kidney, heart and lung of all groups.

Table II. Summary of the Animal Experiment
GroupTreatmentEffective numberBody weight (g)Anti-Hp IgG titer (AI)Serum gastrin (pg/ml)Relative organ weights (%)
LiverKidney
  • Values for results are expressed as means ± SD. Hp, Helicobacter pylori; AI, arbitrary index; CAPE, caffeic acid phenethyl ester.

  • 1

    p < 0.01 vs. Group B–D.

  • 2

    p < 0.01 vs. Group F.

  • 3

    p < 0.05 vs. Group F.

AHp + 0.1% CAPE1081.4 ± 8.718.4 ± 3.12187 ± 52.333.88 ± 0.250.82 ± 0.07
BHp + 0.03% CAPE1073.5 ± 4.811.5 ± 4.22176 ± 35.333.74 ± 0.170.82 ± 0.04
CHp + 0.01% CAPE1072.6 ± 4.215.9 ± 7.32192 ± 93.233.66 ± 0.270.80 ± 0.05
DHp1073.0 ± 6.810.6 ± 5.42185 ± 45.833.74 ± 0.130.83 ± 0.063
EBroth + 0.1% CAPE569.5 ± 4.90.7 ± 0.4126 ± 16.73.59 ± 0.300.73 ± 0.24
FBroth1075.2 ± 6.40.5 ± 0.1125 ± 54.83.75 ± 0.210.77 ± 0.04

Inhibitory effects of CAPE on H. pylori-induced gastritis

The gastric mucosa of H. pylori-infected groups (Groups A–D) was generally thickened and edematous, with occasional erosions and ulceration. Such macroscopic lesions were not recognized in the stomachs of noninfected gerbils, and gastric mucosal specimens from these gerbils had normal histomorphology. Histological findings for chronic gastritis in each group are summarized in Table III. Infiltration of neutrophils in both the antrum and corpus and of mononuclear cells in the antrum of Group A animals (H. pylori + 0.1% CAPE) was significantly suppressed as compared to Group D (p < 0.05 and 0.01, respectively) (Figs. 6a, 6e, and 6i). There were no significant differences in scores for intestinal metaplasia, heterotopic proliferative glands and COX-2 immunoreactivity among Groups A–D. Macroscopic and microscopic analyses revealed no significant differences between gerbils in Groups E and F, so Group E was excluded from subsequent analyses of BrdU labeling indices, immunohistochemistry of NF-κB p50 and phospho-IκB-α and transcriptional expression of inflammatory factors. Immunohistochemistry of NF-κB p50 and phospho-IκB-α revealed that strong reactivity of gastric epithelium and infiltrated cells in H. pylori-infected gerbils, and CAPE treatment significantly reduced the immunohistochemical scores (Figs. 6c, 6d, 6g, 6h, 6k, and 6l).

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Figure 6. Histopathology and immunohistochemistry findings for the gastric mucosa of Mongolian gerbils. Helicobacter pylori (H. pylori) + basal diet group (Group D, panels a–d), H. pylori + 0.1% caffeic acid phenethyl ester (CAPE) group (Group A, panels e–h) and Broth + basal diet group (Group F, panels i–l). (a) Marked mucosal hyperplasia with severe infiltration of neutrophils and mononuclear cells (H&E, ×50, Bar = 200 μm). (b) Large numbers of BrdU-positive cells are apparent in hyperplastic mucosal epithelium (BrdU immunostaining, ×100, Bar = 100 μm). (c and d) Strong expression of NF-κB p50 and phospho-IκB-α is observed in gastric epithelium and infiltrated cells (NF-κB p50 and phospho-IκB-α immunostaining, respectively, ×100, Bar = 100 μm). (e) Mild to moderate gastritis (H&E, ×50, Bar = 200 μm). (f) Note moderate numbers of BrdU-positive cells (BrdU, ×100, Bar = 100 μm). (g and h) Relatively weak expression of NF-κB p50 and phospho-IκB-α in 0.1% CAPE-treated gerbils (NF-κB p50 and phospho-IκB-α, ×100, Bar = 100 μm). (i) No lesions are observed in the gastric mucosa of a noninfected gerbil (H&E, ×50, Bar = 200 μm). (j) BrdU-positive cells are present only in the proliferative zone of a noninfected animal (BrdU, ×100, Bar = 100 μm). (k and l) NF-κB p50 and phospho-IκB-α expression is weak in a noninfected gerbil (NF-κB p50 and phospho-IκB-α, ×100, Bar = 100 μm).

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Table III. Histopathological Evaluation of Gastritis
GroupTreatmentEffective numberInfiltration of neutrophilsInfiltration of mononuclear cellsIntestinal metaplasiaHeterotopic proliferative glandsScore of COX-2 immunohistochemistry
AntrumCorpusAntrumCorpusAntrumCorpusAntrumCorpusAntrumCorpus
  • Values for results are expressed as means ± SD. COX-2, cyclooxygenase-2; Hp, Helicobacter pylori; CAPE, caffeic acid phenethyl ester.

  • 1

    p < 0.05 vs. Group D.

  • 2

    p < 0.01 vs. Group D.

AHp + 0.1% CAPE101.8 ± 0.411.1 ± 0.612.3 ± 0.321.6 ± 0.40.0 ± 0.00.0 ± 0.01.1 ± 0.40.6 ± 0.41.3 ± 0.50.9 ± 0.7
BHp + 0.03% CAPE102.6 ± 0.52.1 ± 0.82.9 ± 0.22.3 ± 0.50.1 ± 0.30.2 ± 0.41.3 ± 0.51.0 ± 0.71.6 ± 0.51.3 ± 0.8
CHp + 0.01% CAPE102.1 ± 0.42.0 ± 0.92.6 ± 0.52.2 ± 0.60.1 ± 0.30.1 ± 0.31.1 ± 0.40.9 ± 0.41.4 ± 0.50.9 ± 0.6
DHp102.5 ± 0.52.2 ± 0.72.9 ± 0.22.2 ± 0.60.1 ± 0.30.1 ± 0.31.1 ± 0.50.9 ± 0.71.7 ± 0.71.2 ± 0.8
EBroth + 0.1% CAPE50.0 ± 0.00.1 ± 0.20.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.00.2 ± 0.40.2 ± 0.4
FBroth100.1 ± 0.20.0 ± 0.00.2 ± 0.20.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.00.1 ± 0.30.0 ± 0.0

BrdU labeling indices in gastric epithelial cells

In H. pylori-infected gerbils, BrdU-labeled epithelial nuclei were found distributed throughout the hyperplastic mucosa, while BrdU-positive cells in noninfected animals were located in the neck portions of glands (Figs. 6b, 6f, and 6j). At 12 weeks, BrdU labeling indices in both the antrum and corpus of Group A (H. pylori + 0.1% CAPE) were significantly suppressed as compared to Group D (p < 0.01; Fig. 7). Similarly, BrdU labeling indices in the antrum of the 0.03% CAPE-treated group (Group B) were significantly lowered (p < 0.05), without significant decrease in the corpus.

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Figure 7. Immunohistochemical analysis of BrdU, NF-κB p50, and phospho-IκB-α staining in the antrum and corpus of Mongolian gerbils. BrdU staining was evaluated by mean percentages of BrdU-positive cells among total cells in 10 selected glands, and NF-κB p50 and phospho-IκB-α staining were scored 0–4. Data are presented as mean ± SD values. *p < 0.05 and **p < 0.01 vs. Group D.

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Hyperplasia in isolated pyloric glands

To evaluate the effect of CAPE on H. pylori-induced mucosal hyperplasia, we analyzed the length of isolated glands from the pyloric mucosa (Fig. 8a). The average value for Group A (H. pylori + 0.1% CAPE) was significantly reduced compared to that for Group D (p < 0.05) (Fig. 8b).

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Figure 8. Isolated glands from the pyloric mucosa of Mongolian gerbils. (a) Histopathology and immunohistochemistry of isolated pyloric glands (Upper column, H&E; Lower column, BrdU staining, ×200, Bar = 25 μm). Note the Helicobacter pylori-induced hyperplasia and cell proliferation activity is attenuated by 0.1% CAPE treatment. (b) Average lengths of isolated pyloric glands of Mongolian gerbils. Data are mean ± SD values. Note that the Y-axis starts from 150. *p < 0.05 vs. Group D.

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Expression of inflammatory factors in the pyloric mucosa

RT-PCR data for mRNA expression of inflammatory cytokines and enzymes in the pyloric mucosa of gerbils are summarized in Figure 9. TNF-α and iNOS mRNA expression in Group A (H. pylori + 0.1% CAPE) was significantly suppressed as compared to Group D (p < 0.05). Levels of IL-2 mRNA in Groups A and B and IFN-γ and IL-6 mRNAs in all CAPE-treated groups (Groups A–C) were also markedly lower than in Group D (p < 0.05). Only very low mRNA expression was evident in Group F.

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Figure 9. Relative expression levels of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-2, IL-6, IL-10, inducible nitric oxide synthase (iNOS), and KC mRNAs in the pyloric mucosa of Mongolian gerbils at 12 weeks postinfection. Values were set at 1.0 in Group F and expressed as mean ± SE relative values. Note that the Y-axes have different scales. *p < 0.05 vs. Group D.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In this study, we demonstrated that NF-κB transcriptional activation in H. pylori-stimulated AGS gastric cancer cells were significantly inhibited by CAPE treatment in a dose-dependent manner. This result is consistent with a previous report that CAPE inhibits H. pylori-induced DNA-binding activity of NF-κB in gastric cancer cells.9 We found that CAPE treatment resulted in a decrease of the phosphorylation of NF-κB p65 subunit and inhibition of IκB-α degradation. In addition, relative quantitative real-time RT-PCR analysis revealed that mRNA expressions of several inflammatory factors including IL-1β, TNF-α, iNOS, and IL-8 in NF-κB-activated AGS cells were significantly suppressed by CAPE treatment in a dose-dependent manner, whereas there were no statistical differences in COX-2 and IL-10 expression. IκB-α degradation induces the phosphorylation of p65 and following nuclear transition of NF-κB complex. Thus, our data suggested that CAPE may prevent H. pylori-induced NF-κB activation and transcriptional activity of inflammatory factors through the inhibition of nuclear translocation of NF-κB.

Here we found that oral administration of CAPE effectively inhibited gastric inflammation at 0.1% in the diet with significant suppression of infiltration of neutrophils both in the antrum and corpus and mononuclear cells in the antrum at week 12. Fitzpatrick et al. earlier reported CAPE to inhibit TNF-α production in a rat macrophage cell line and TNF-α-stimulated IL-8 production in human colonic epithelial cells.36 Expression of IL-8, a potent chemokine stimulus for neutrophil migration, is increased with H. pylori infection through NF-κB activation.17 In this study, mRNA expression of TNF-α and IL-8 in H. pylori-stimulated AGS cells were significantly decreased by CAPE treatment. Similarly, CAPE markedly suppressed the expression of TNF-α and KC protein, one of the IL-8 homologues, in H. pylori-infected gerbils. The inhibitory effects of CAPE on infiltration of neutrophils observed in this study may therefore be explained by reduction of NF-κB-associated cytokines, including TNF-α and KC.

Phosphorylation of IκB-α acts as a trigger of IκB degradation, allowing the nuclear translocation of NF-κB complex and activation of gene expression. In our study, 0.1% CAPE-containing diet inhibited the immunohistochemical expression of phosphorylated IκB-α and nuclear transition of NF-κB p50 subunit in the gastric mucosa of H. pylori-infected gerbils, suggesting that CAPE has chemopreventive potentials by inhibiting the NF-κB pathway.

Our findings for BrdU-labeled cells in the gastric mucosa and average lengths of isolated pyloric glands suggest that H. pylori-induced chronic gastritis and epithelial hyper-proliferation are efficiently suppressed by CAPE administration. Previous clinical studies have demonstrated that epithelial proliferation is positively correlated with the degree of histological inflammation in the gastric mucosa of H. pylori-infected patients.37 We have previously reported that the severity of gastritis plays an important role in H. pylori-associated gastric carcinogenesis in gerbils, with essential involvement of chronic inflammation and increased rates of cell proliferation.38 Therefore, it is very conceivable that CAPE might reduce gastric carcinogenesis as well as chronic gastritis.

Our demonstration that mRNA expression levels of inflammatory factors including TNF-α, IFN-γ, IL-2, IL-6, iNOS and KC were significantly decreased by CAPE treatment in the pyloric mucosa of H. pylori-infected Mongolian gerbils is of clear interest. All these factors are known to be induced by NF-κB transcriptional activation.5, 39 It has been reported that a predominant H. pylori-specific Th1 response characterized by TNF-α and IFN-γ production is associated with H. pylori-infected gastritis.40 Several studies have demonstrated that IFN-γ and IL-6 play important roles in progression of pyloric gastritis in H. pylori-infected gerbils.20, 33 In addition, TNF-α is a mediator during inflammation and tumor promotion, leading to activation of NF-κB and thereby suppression of cell death and stimulation of cell proliferation.41 Interestingly, although significant suppressive effects of CAPE on IL-6 and IFN-γ expression in the antrum were observed in all CAPE-treated groups, gastritis was attenuated only in 0.1% CAPE-treated gerbils. Thus, our results suggest that TNF-α and iNOS might be key molecules, in addition to other factors, suppressing H. pylori-induced chronic gastritis in Mongolian gerbils. iNOS is also known to be up-regulated by H. pylori and to enhance progression of gastric inflammation and carcinogenesis through generation of reactive oxygen species.42

On the other hand, expression level of COX-2 was not suppressed by CAPE treatment both in H. pylori-stimulated AGS cells and in the pyloric mucosa of gerbils. Several studies demonstrated that gastric COX-2 expression in H. pylori-infected humans and rodents could be associated with repair of mucosal injury.43–45 Thus, there is a possibility that COX-2 expression may play important roles both in mediation of gastritis and in healing of H. pylori-associated gastric ulceration. CAPE treatment also showed no effects on mRNA expression of IL-10 in AGS cells and the pyloric mucosa of gerbils. IL-10 has been known as an anti-inflammatory cytokine, and demonstrated to inhibit other inflammatory cytokines and chemokines in H. pylori-induced gastritis.46, 47 Our result of stable expression of IL-10 may reflect an increase of anti-inflammatory cytokine activity through the separate cascade from NF-κB pathway.

CAPE concentrations in the range of 0.01–0.1% were chosen for the present investigation because a previous study in mice demonstrated no toxicity at 0.15% CAPE given for 110 days.11 In this study, there were no significant differences in relative organ weights of liver and kidney between 0.1% CAPE-treated (Group E) and nontreated gerbils (Group F). In addition, no macroscopic and microscopic lesions were observed in the nongastric internal organs, including liver, spleen, kidney, heart and lung of CAPE-treated gerbils. Therefore, we conclude that the toxicity of CAPE is negligible at the doses used in the present study. Our data showed that relative kidney weight of H. pylori-infected group (Group D) was statistically higher than that of Group F. Several authors have discussed whether H. pylori infection is associated with the pathogenesis of renal failures in humans, but the relationship is still unclear.48, 49 Because there were no macroscopic and microscopic renal lesions in the gerbils, more detail examination is needed to clarify the association of renal weight change and H. pylori infection. Regarding body weight, 0.1% CAPE-treated and H. pylori-infected gerbils (Group A) were heavier than those in other infected groups (Groups B–D). However, animals in Group E showed no significant increase. Since there were no marked differences in serum total cholesterol and triglyceride levels between Groups A and D (data not shown), there may not be significant relevance between CAPE ingestion and body weight change.

In conclusion, our study clearly demonstrated: (1) CAPE treatment inhibits H. pylori-induced NF-κB activation by suppression of IκB-α degradation and phosphorylation of p65 in a gastric cancer cell line, and (2) CAPE exerts anti-inflammatory effects on H. pylori-induced gastritis in Mongolian gerbils with reduction of nuclear transition of NF-κB p50 through phosphorylation of IκB-α and suppression of the mRNA expression of many NF-κB-associated inflammatory factors. These results suggest that CAPE may have potential as an alternative drug for chemoprevention of chronic active gastritis and other H. pylori-associated gastric disorders, including stomach adenocarcinomas.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Ms. Noriko Saito and Ms. Ayumi Saito for their expert technical assistance.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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