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Keywords:

  • anthocyanin;
  • Helicobacter pylori;
  • interleukin-8;
  • reactive oxygen species

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. DISCLOSURE
  8. REFERENCES

Infection with Helicobacter pylori leads to gastritis, peptic ulcers and gastric cancer. Moreover, when the gastric mucosa is exposed to H. pylori, gastric mucosal inflammatory cytokine interleukin-8 (Il-8) and reactive oxygen species increase. Anthocyanins have anti-oxidative, antibacterial and anti-inflammatory properties. However, the effect of anthocyanins in H. pylori-infected cells is not yet clear. In this study, therefore, the effect of anthocyanins on H. pylori-infected human gastric epithelial cells was examined. AGS cells were pretreated with anthocyanins for 24 hrs followed by H. pylori 26695 infection for up to 24 hrs. Cell viability and ROS production were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 2′,7′–dichlorofluorescein diacetate assay, respectively. Western blot analyses and RT-PCR were performed to assess gene and protein expression, respectively. IL-8 secretion in AGS cells was measured by ELISA. It was found that anthocyanins decrease H. pylori-induced ROS enhancement. Anthocyanins also inhibited phosphorylation of mitogen-activated protein kinases, translocation of nuclear factor-kappa B and Iκβα degradation. Furthermore anthocyanins inhibited H. pylori-induced inducible nitric oxide synthases and cyclooxygenase-2 mRNA expression and inhibited IL-8 production by 45.8%. Based on the above findings, anthocyanins might have an anti-inflammatory effect in H. pylori-infected gastric epithelial cells.

List of Abbreviations
COX-2

cyclooxygenase-2

DCF-DA

2′,7′–dichlorofluorescein diacetate

ERK

extracellular signal-regulated kinase

H. pylori

Helicobacter pylori

IL

interleukin

iNOS

inducible nitric oxide synthases

JNK

c-Jun N-terminal kinase

MAPK

mitogen-activated protein kinase

WST-1

water soluble tetrazolium salts-1

NAC

N-acetyl-cysteine

NF-κB

nuclear factor-kappa B

ROS

reactive oxygen species

TBST

Tris-buffered saline containing 0.05% Tween 20

UV

ultraviolet

Helicobacter pylori infection induces inflammation in gastric epithelial cells; this may play a significant role in the pathological events associated with infection by this organism [1, 2]. H. pylori induce inflammatory-associated gene expression in gastric epithelial cells, including that for ROS, COX-2, iNOS, and IL-8 [3-7]. ROS cause oxidative damage to DNA, proteins and lipids. Production of excessive ROS/reactive nitrogen species reportedly occurs in H. pylori-infected human gastric mucosa and, because of the resultant serious imbalance between production of ROS/reactive nitrogen species and antioxidant defense, the amount of ROS correlates with the degree of histological damage in the gastric mucosa. Oxidative stress caused by H. pylori contributes to inflammation, apoptosis and carcinogenesis [8].

Reactive oxygen species induced by H. pylori stimulate MAPKs, such as ERK, JNK and p38, and up-regulate transcription of NF-κB. NF-κB is a crucial regulator of many cellular processes, including control of the immune response and inflammation. Co-culture of H. pylori with cells can induce IL-8 via activation of the transcriptional regulator NF-κB. IL-8, a potent neutrophil-activating chemokine, is central to the immunopathogenesis of H. pylori-induced tissue injury [9].

Anthocyanins, polyphenols derived from many of the colors of fruit and flowers [10], are novel, safe, proven antioxidants and chemopreventive agents [11, 12]. Their basic skeletons are made up of 2-phenylbenzopyrylium or flavylium glycoside. These pigments act as powerful antioxidants that help to protect plants from various factors such as UV light damage. A broad spectrum of in vitro and in vivo studies has demonstrated that anthocyanins promote antioxidant status, healthy vision and anti-angiogenic, antibacterial and anti-inflammatory properties [10]. Anthocyanins also inhibit the growth of some cancer cells [13]. Anthocyanins are especially abundant in the epidermis palisade layer of the black soybean seed coat, which contains cyanidin-3-glucoside, delphinidin-3-glucoside and petunidin-3-glucoside [10, 14-17]. Cyanidin-3-glucoside is reportedly an effective antioxidant that helps prevent cerebrovascular and cardiac disorders; in this regard it is superior to other components of anthocyanins [18]. Although many studies have demonstrated the beneficial effects of anthocyanins on antioxidants, the anti-inflammation mechanism has not yet been clarified. Recently, Kim et al. reported that anthocyanins inhibit secretion of H. pylori CagA and VacA [19].

The goal of this study was to determine the anti-inflammatory effects of anthocyanins against experimental H. pylori infection. In order to increase their clinical potential in gastrointestinal disease, we also hoped to clarify the mechanism of their protective effects against H. pylori-induced inflammation.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. DISCLOSURE
  8. REFERENCES

Bacterial strains and culture conditions

The H. pylori strain 26695 was supplied by the H. pylori Korean Type Culture Collection (Gyeongsang National University School of Medicine, Gyeongsang, Korea). H. pylori was grown and maintained on brucella agar (Difco, Detroit, MI, USA) supplemented with 10% bovine serum (Lonza, Walkersville, MD, USA) in 10% CO2 at 37°C and 100% humidity in a CO2 incubator.

Anthocyanin extraction and purification

Anthocyanins were extracted from black soybean (Glycine max [L.] Merr) and purified as described by Kim et al. [14]. The compositions of anthocyanins, determined by the peak area ratio on high-performance liquid chromatography, consisted of cyanidin-3-glucoside (72%), delphinidin-3-glucoside (20%) and petunidin-3-glucoside (6%). No contaminants were detected.

Cell culture

A human gastric epithelial cell line, AGS (gastric adenocarcinoma, ATCC CRL 1739), was purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were grown in complete medium, consisting of RPMI-1640 medium (Lonza, Walkersville, MD, USA) supplemented with 10% FBS. The cells were pretreated with different amounts of anthocyanins for 24 hrs, washed three times with PBS and then infected with H. pylori in complete medium at a multiplicity of infection of 200:1.

Determination of cell viability

Cell viability was determined by the tetrazolium salt reduction method using WST-1 according to the manufacturer's instructions (BioVision, Mountain View, CA, USA). In brief, 2 × 104 cells/well were plated in 96-well plates and cultured overnight. The cells were incubated with/without anthocyanins for 24 hrs, after which 10 µL of WST-1 was added and the cells incubated for an additional 1 hr. The number of viable cells was estimated by measuring the absorbance at 450 nm. All treatments were performed in quadruplicate and the experiment was repeated three times. Cell viability was calculated as the relative absorbance compared with that of control cultures.

Measurement of intracellular reactive oxygen species

Intracellular ROS production was measured by DCF-DA (Sigma, St. Louis, MO, USA) using confocal laser scanning microscopy. Anthocyanins or NAC were pretreated as mentioned above. Cells were infected with H. pylori for 24 hrs. After 2 hrs, dishes of sub-confluent cells were washed with PBS and incubated in the dark for 5 mins in the presence of 5 µM 2′,7′-DCF-DA. After being washed, the culture dishes were transferred to an inverted microscope with a confocal imaging system (Olympus, Tokyo, Japan), and ROS generation detected by oxidation of DCF-DA using a fluorescein isothiocyanate filter set. The green color represents ROS; the fluorescence density was normalized to cell count. Pixel images were collected by single rapid scans. Fluorescent images were analyzed using FV10-ASW software. Analyses were repeated three times over the same region.

Western blot analysis

Three × 106 cells were homogenized in 150 µL of cell lysis buffer (Pro-prep, Intron, Biotechnology, Sungnam, Korea). The cytoplasmic protein fraction was separated using a Nuclear/Cytosol Fractionation Kit (BioVision, Mountain View, CA, USA). Protein concentrations were determined by the Lowry assay. Equal quantities of protein (20 µg) were loaded per lane, separated by SDS–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA) for 1 hr at 20 V with an SD semidry Transfer Cell (Bio-Rad Laboratories). The membranes were blocked with 5% skimmed milk in TBST for 2 hrs at room temperature. The membranes were then incubated with primary antibodies (anti-Iκβα, anti-p-Iκβα, anti-NF-κB, anti-MAPK antibodies) at a dilution of 1:1000 in TBST overnight at 4°C. Bound antibody was detected by horseradish peroxidase-conjugated secondary antibodies and signals were detected by the enhanced chemiluminescence method (GE Healthcare, Piscataway, NJ, USA).

Reverse transcription polymerase chain reaction analysis for inducible nitric oxide synthases and cyclooxygenase-2

The gene expression of COX-2 and iNOS mRNA was assessed using RT-PCR standardized with the housekeeping gene β-actin. Total RNA was isolated from the cells with the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Total RNA was reverse transcribed into cDNA and used for PCR with human specific primers for COX-2, iNOS and β-actin. The PCR primers are presented in Table 1. Briefly, the products were amplified by 25–30 repeated cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 30 s. During the first cycle, the 95°C step was extended to 5 mins and on the final cycle, the 72°C step was extended to 5 mins. The PCR products were separated on 1.5% agarose gels containing 0.5 µg/mL ethidium bromide and visualized by UV transillumination [20].

Table 1. Primers used for RT-PCR
GenesPrimer sequencesProduct size (bp)Ref.
COX-25′-TTCAAATGAGATTGTGGGAAAATTGCT-3′305[20]
 5′-AGATCATCTCTGCCTGAGTATCTT-3′  
iNOS5′-CGGTGCTGTATTTCCTTACGAGGCGACGAAGG-3′259[20]
 5′-GTGCTGCT TGT TAGGAGGTCAAGTAAAGGGC-3′  
β-Actin5′-ACCAACTGG GACGACATGGAG-3′353[20]
 5′-GTGAGG ATCTTCA TGAGGTAGTC-3′  

Determination of interleukin-8

AGS cells (2 × 104 cells) were seeded in 96-well plates. The cells were pretreated with anthocyanins (12.5, 25 and 50 µg/mL) or 20 mM NAC (3264 µg/mL) for 24 hr [21]. The cells were then infected with H. pylori for 24 hrs. The culture medium was collected, and IL-8 measurements performed using ELISA kits (Enzo Life Sciences, Farmingdale, NY, USA). Each sample was tested in triplicate.

Statistical analysis

All samples were analyzed in triplicate. Data are presented as the mean ± SD. SPSS (SPSS, Chicago, IL, USA) was used to test statistical significance by ANOVA. A value of P < 0.05 was considered to be statistically significant.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. DISCLOSURE
  8. REFERENCES

Cytotoxicity of anthocyanins in AGS cells

It was confirmed that exposure to 50 µg/mL of anthocyanins had no significant effect on cell proliferation at the concentrations examined (12.5–200 µg/mL) (Fig. 1). It was therefore concluded that anthocyanins at these concentrations had no cytotoxic effects on AGS cells and could be used in this study.

image

Figure 1. Effect of anthocyanins on the proliferation of AGS cells. AGS cells were incubated with the indicated concentrations of anthocyanins. Cell viability was determined by an WST-1 assay. Data are presented as the mean ± SD. Statistical assessments were performed by Student's t-test. *, P < 0.05, significant compared to control cells.

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Anthocyanins decrease Helicobacter pylori-induced reactive oxygen species production in AGS cells

Using a DCF-DA fluorescence dye, confocal microscopic observation showed increased amounts of ROS in AGS cells infected with H. pylori. Increases in ROS induced by H. pylori were inhibited by treatment with anthocyanins in a dose-dependent manner. Treatment with anthocyanins (12.5, 25 and 50 µg/mL) significantly reduced the amount of ROS in H. pylori-infected AGS cells (Fig. 2).

image

Figure 2. Effect of anthocyanins on ROS production induced by H. pylori in AGS cells. Cells were treated as described in the Materials and Methods Section. (a) Uninfected cells. (b) H. pylori-infected cells. (c) 1 mM of H2O2-treated cells. (d) Cells pretreated with NAC. (e) H. pylori-infected cells pretreated with NAC. (F–H) H. pylori-infected cells pretreated with 12.5, 25 and 50 µg/mL of anthocyanins. Data for the lower panel of this figure were created by the equation number of green cells/total cells × 100.Statistical assessments were performed by Student's t-test. *, P < 0.05, significant compared to H. pylori-infection.

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Anthocyanins inhibit Helicobacter pylori-activated mitogen-activated protein kinases in AGS cells

After 30 mins of stimulation by H. pylori, MAPK activation was determined by measuring both the phospho-specific and full forms of ERK, JNK and p38. The amounts of full forms of ERK, JNK and p38 in AGS cells were not changed by H. pylori treatment; however, H. pylori phosphorylated ERK, JNK and p38. Anthocyanins inhibited phosphorylation of three MAPKs (ERK, JNK and p38) induced by H. pylori in a dose-dependent manner. However, the total forms of these MAPKs were not affected by anthocyanin treatment (Fig. 3).

image

Figure 3. Immunodetection of ERK, JNK and p38 in H. pylori-infected cells with/without anthocyanins. Cells were treated as described in the Materials and Methods. The amounts of phospho-specific and total forms of MAPKs (ERK, JNK and p38) were determined by western blotting after 30 mins culture. Statistical assessments were performed by Student's t-test. *, P < 0.05, significant compared to H. pylori-infection.

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Anthocyanins inhibit Helicobacter pylori-induced protein expression of phosphorylated-Iκβα and decreased cytosolic nuclear factor-kappa B in AGS cells

To determine if NF-κB activated by H. pylori is inhibited by treatment with anthocyanins, an NF-κB translocation experiment was performed. H. pylori caused NF-κB translocation from the cytosol to the nucleus. This effect was inhibited by pretreatment with anthocyanins in a dose-dependent manner. Phosphorylation by kinases of Iκβα, an inhibitor of NF-κB activity, resulted in degradation of Iκβα and the subsequent release of NF-κB, which translocates to the nucleus where it is active in regulation of gene transcription. Thus, it was determined that if anthocyanins prevent phosphorylation of Iκβα by H. pylori, H. pylori induces Iκβα phosphorylation, which is prevented by anthocyanins in a dose-dependent manner (Fig. 4a).

image

Figure 4. Effect of anthocyanins on H. pylori-induced inflammation. (a) Immunodetection of Iκβα, p-Iκβα, cytosolic NF-κB and COX-2 in H. pylori-infected cells with/without anthocyanins. After 6 hrs incubation with H. pylori, cells were treated as described in the Materials and Methods Section. The results were confirmed by three independent experiments. (b) Effect of anthocyanins on H. pylori-induced expression of iNOS and COX-2 in AGS cells. Cells were treated as described in Materials and Methods Section. After 6 hrs incubation, they were assessed by RT-PCR. Statistical assessments were performed by Student's t-test. *, P < 0.05, significant compared to H. pylori-infection.

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Anthocyanins inhibit Helicobacter pylori-induced mRNA expression of cyclooxygenase-2 and inducible nitric oxide synthases

After 6 hrs of infection with H. pylori, mRNA expression of COX-2 and iNOS in AGS cells increased significantly according to RT-PCR analysis. However, in AGS cells anthocyanins inhibited H. pylori-stimulated mRNA expression of COX-2 and iNOS in a concentration-dependent manner (Fig. 4b).

Anthocyanins decrease Helicobacter pylori-induced interleukin-8 production in AGS cells

The effect of anthocyanins on H. pylori-induced IL-8 production by gastric epithelial cells was examined. IL-8 production in gastric epithelial cells infected with H. pylori with or without pre-incubation of anthocyanins from 12.5 to 50 µg/mL was measured. Pretreatment of H. pylori-infected AGS cells with 12.5, 25 and 50 µg/mL of anthocyanins for 24 hrs significantly decreased IL-8 production by 77.2%, 71.3% and 68.8%, respectively, compared to H. pylori-infected cells without anthocyanins (Fig. 5). To test whether this phenomenon is caused by ROS-induced production, cells were pre-incubated with 3264 µg/mL NAC before being infected with H. pylori. A clear decrease in IL-8 production was found.

image

Figure 5. Anthocyanins inhibit IL-8 release from H. pylori-infected AGS cells. Cells were treated as described in the Materials and Methods Section. The results were confirmed by three independent experiments. Statistical assessments were performed by Student's t-test. *, P < 0.05, significant compared to H. pylori-infection.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. DISCLOSURE
  8. REFERENCES

In the present study, we investigated the effects on the responsiveness of H. pylori infected-human gastric epithelial cells of anthocyanins from black soybean (Glycine max [L.] Merr). This potential effect of anthocyanins was investigated by exploring various variables that are usually associated with inflammation, namely, (i) increase of ROS upon stimulation with H. pylori; (ii) phosphorylation of three MAPKs: ERK, JNK and p38; (iii) modulation of NF-κB and Iκβα activity; (iv) degree of expression of COX-2 and iNOS; and (v) secretion of the pro-inflammatory cytokine IL-8.

It has been shown that H. pylori induces increased ROS, MAPK and NF-κB activation in AGS cells [4, 9, 22]. ROS may also play a role as the initial molecules in the signaling and regulation of gene expression associated with the biological effects elicited by inflammation. Anthocyanins can alleviate these effects by their antioxidant actions and by modulating the downstream pathway. Our experiments showed that pretreatment of AGS cells with anthocyanins decreases ROS production.

Helicobacter pylori-induced ROS generation can also activate diverse downstream signaling molecules, such as MAPK. Lin et al. reported that ROS and inflammatory cytokines were associated with MAPK activation [23]. MAPK signaling is one of the most important signaling pathways in immune responses. Numerous researchers have reported that H. pylori up-regulates NF-κB activity through the MAPK signaling pathway [24-28]. Indeed, H. pylori phosphorylates all three types of MAPK (ERK, JNK and p38). Moreover, H. pylori activates NF-κB and p-Iκβα in AGS cells. However, anthocyanins suppress H. pylori-induced ROS expression and inhibit successive MAPK signaling.

Nuclear factor-kappa B is an oxidant-sensitive transcriptional factor and ROS have an important role in its activation in H. pylori-infected AGS cells [21]. It has previously been shown that H. pylori decreases cytosolic amounts of NF-κB and increases p-Iκβα in AGS cells [7, 29]. In response to various inflammatory stimuli, Iκβα is degraded and the NF-κB complex migrates into the nucleus and binds DNA recognition sites in the regulatory regions of target genes, including those for iNOS and COX-2 [5, 30]. All of the activation mechanisms that lead to NF-κB translocation may involve ROS [31, 32]. These findings suggest that, under the influence of ROS, activated MAPK augments activation of NF-κB and subsequent transcription of COX-2, and that iNOS is mediated by activation of NF-κB in H. pylori-infected AGS cells.

In this study, anthocyanins did decrease activation of MAPK, NF-κB, iNOS and COX-2 according to western blot analysis and RT-PCR. Being powerful antioxidants [17], anthocyanins affect inflammation by blocking ROS at the initial step and regulating the NF-κB and MAPK signaling pathways activated by H. pylori. According to the present results, anthocyanins simultaneously inhibit activation by H. pylori of all three MAPKs. The definitive one of these three types of MAPK signal pathway is not yet clear; however, there is definitely signal interactions among the MAPKs [33].

Antioxidants such as curcumin and β-carotene reportedly inactivate NF-κB by inhibiting phosphorylation of Iκβα [32, 34]. Thus, we investigated whether anthocyanins can prevent Iκβα phosphorylation and found that anthocyanins reduce H. pylori-induced phosphorylation of Iκβα in a dose-dependent manner. Therefore, the prevention by anthocyanins of Iκβα decrease indicates inhibition of NF-κB activation by H. pylori in AGS cells.

Because IL-8 secreted by gastric epithelial cells is likely to be an important host mediator of induction of neutrophil migration into infection sites, it may be important in regulation of the inflammatory and immune processes in response to H. pylori [35]. NF-κB has a dominant role in H. pylori-induced IL-8 production in gastric epithelial cells [6]. In this study, we found that anthocyanins inhibit H. pylori-induced IL-8 secretion, thus augmenting their benefit in regard to protection of gastric epithelial cells.

In summary, this study shows that anthocyanins from black soybean have both antioxidant effects and the ability to down-regulate ROS generation and decrease the activation of MAPKs, NF-κB, iNOS and COX-2 that is induced by H. pylori (Fig. 6). In addition, anthocyanins decrease production of H. pylori-induced cytokines such as IL-8, suggesting that they may prevent gastric damage. As a consequence, anthocyanins can reduce H. pylori-induced inflammation in gastric epithelial cells.

image

Figure 6. Anthocyanins from black soybean inhibit Helicobacter pylori-induced ROS in human gastric epithelial AGS cells.

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ACKNOWLEDGEMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. DISCLOSURE
  8. REFERENCES

This study was supported by a grant from the National Research and Development Program for Cancer Control, Ministry for Health, and Welfare and Family Affairs of Korea (0820050).

DISCLOSURE

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. DISCLOSURE
  8. REFERENCES

The authors have no conflict of interest to declare.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. DISCLOSURE
  8. REFERENCES
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