Effect of Rumex Aquaticus Herba Extract Against Helicobacter pylori -Induced Inflammation in Gastric Epithelial Cells

Abstract

The purposes of this study were to examine the characteristics of Helicobacter pylori and the effect of Rumex Aquaticus Herba extract on the expression of cytokines in H. pylori-infected gastric epithelial cells. Cultured human adenocarcinoma gastric cells (AGS) were infected by H. pylori in RPMI 1640 media. Cell growth was measured by trypan blue assay. Western blot analysis was performed to investigate effect of extract containing Quercetin-3-O-β-d-glucuronopyranoside (ECQ) on the expression of inflammatory factors and the inhibition on cell growth. Furthermore, we compared the inhibitory effects with various combinations of clarithromycin, amoxicillin, omeprazole, and ECQ. The urease test with Christensen's Urea Agar was performed to identify the urease activity of H. pylori and the effect ECQ has on urease activity. When the cells were exposed to H. pylori, the trypan blue assay revealed a decrease in the rate of cell growth. Western blot analysis showed that H. pylori-infected cells had increased levels of degraded IκB-α and inflammatory factors. Pretreatment with ECQ inhibited interleukin expression induced by H. pylori in a dose-dependent manner. A combination of ECQ and antibiotics inhibited cytokine expression more effectively than other treatments. H. pylori displayed significant urease activity. ECQ did not significantly inhibit urease activity. These data suggest that H. pylori infection has cytotoxic effects against AGS cells, and ECQ may inhibit the production of proinflammatory cytokines in H. pylori-infected AGS cells.

Introduction

H elicobacter Pylori (H. pylori) is a Gram-negative, spiral-shaped microaerophilic bacterium, which is involved in the pathogenesis of chronic gastritis, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma.¹ H. pylori infection increases apoptosis of epithelial cell in the gastric mucosa, which may play a pivotal role in gastric carcinogenesis.² The presence of a pathogenicity island determines the pathogenicity of H. pylori strains and PAI encodes the immunodominant cytotoxin-associated antigen (CagA).³ H. pylori adheres to the host cell to translocate CagA into the cell body. When PAI-positive H. pylori invades the host cells, the ubiquitous transcription factor NF-κB and related signaling pathways are activated.⁴ The activity of NF-κB includes various immune responses and promotes inflammation, which is tightly regulated by interaction with inhibitory IκB. IκB-α forms a complex with NF-κB in the cytosol to inhibit the nuclear translocation of NF-κB, which is activated after the phosphorylation of IκB-α.^5
–7 As one of the major proinflammatory cytokines, IL-8 is an important target of NF-κB.⁸ IL-8 plays a major role in the initiation and maintenance of inflammation, and also has been identified as a carcinogenic factor.⁸ Because IL-8 is established as a neutrophil-activating chemokine, secretion of IL-8 by infected gastric epithelial cells may be important in regulating neutrophil infiltration to the gastric mucosa in H. pylori gastritis. Moreover, increased expressions of IL-1α and IL-1β were seen in the gastric mucosa of patients infected with CagA-positive H. pylori. ⁹ The most widely used therapy for H. pylori infection is a 1 week triple therapy with a proton pump inhibitor, such as omeprazole, and the antibiotics clarithromycin and amoxicillin. However, this regimen has various side effects and is ineffective in 20–30% of patients.¹⁰

Rumex Aquaticus is a member of the Polygonaceae family, and this plant has been used to treat infestation, diarrhea, edema, jaundice, and constipation in traditional oriental medicine. Quercetin-3-O-β-d-glucuronopyranoside (QGC) is the active ingredient of Rumex Aquaticus and a quercetin derivative. Quercetin (3,5,7,3′4′-pentahydroxyflavone) is a potent natural antioxidant that prevents gastric mucosal lesions produced by ethanol.¹¹ Gastric mucosal injuries induced by HCl were also prevented by quercetin.¹² QGC is more effective than quercetin against experimental reflux esophagitis.¹³ The extract containing QGC (ECQ) was extracted from Rumex Aquaticus Herba by ethanol. In our previous study, ECQ was protective against indomethacin- and ethanol-induced acute gastric damage.¹⁴ In this study, we investigated H. pylori-induced inflammatory mechanisms through various proinflammatory cytokines and examined the protective effect of ECQ against H. pylori-induced gastritis by determining adenocarcinoma gastric cell line (AGS) proliferation and the expression levels of proinflammatory factors. Additional experiments to measure the level of cytokine inhibition were also performed with various combinations of ECQ and widely used drugs.

Materials and Methods

Reagents

ECQ was kindly provided by Prof. W.K. Whang (Pharmacal Botany Resources Laboratory, Chung-Ang University College of Pharmacy, Seoul, Korea). ECQ was extracted from Rumex Aquaticus with ethanol containing 10.7% QGC, of which the chemical structure was shown in previous study.¹⁵ Bovine serum albumin, ethylene glycol-bis-(β-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA), ethylenediamine tetra acetic acid (EDTA), trypan blue solution (0.4%), leupeptin, aprotinin, β-mercaptoethanol, phenylmethylsulfonylfluoride (PMSF), triton X-100, sodium dodecyl sulfate (SDS), hank's balanced salt solution-modified (HBSS), clarithromycin, omeprazole, amoxicillin, christensen's urea agar, and urea were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS), antibiotic-antimycotic (penicillin, streptomycin, and amphotericin B), and trypsin-EDTA were purchased from Invitrogen (Grand Island, NY, USA). RPMI 1640, tris-buffered saline, and phosphate-buffered saline (PBS) were purchased from Welgene, Inc. (Daegu, Korea). Anti-IκB-α, anti-IL-8, and anti-IL-1β antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); goat anti-rabbit IgG-HRP, goat anti-mouse IgG-HRP, and rabbit anti-goat IgG-HRP from Zymed Laboratories, Inc. (Eccles Avenue, CA, USA). The rainbow prestained molecular weight marker was purchased from Elpis Biotechnology (Daejeon, Korea). Enhanced chemiluminescence agents were purchased from PerkinElmer Life Sciences (Boston, MA, USA). Ammonium persulfate, N,N,N’,N’-tetramethylethylendiamine (TEMED), nitrocellulose membrane, tris/glycine/SDS buffer, tris/glycine buffer, restore TM western blot stripping buffer, and 30% acrylamide/bis solution were purchased from BioRad (Richmond, CA, USA).

Bacterial strain

An H. pylori strain (HP99) was isolated from gastric antral mucosa obtained from a Korean patient with a duodenal ulcer and was identified as CagA+ and VacA+.¹⁶ HP99 was kindly supplied by Dr. J.G. Kim (Chung Ang University College of Medicine, Seoul, Korea). Bacteria were inoculated onto blood agar plates (Hanil Komed, Songnam, Korea) at 37°C under microaerophilic conditions using a 10% CO₂ incubator (Vision Scientific, Bucheon, Korea).

Cell culture and H. pylori stimulation

The human gastric epithelial cell line AGS (gastric adenocarcinoma, KCLB 21739) was obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were grown in complete medium, consisting of RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 μg/mL amphotericin B. AGS cells were seeded and cultured to reach 80% confluency. H. pylori was harvested, washed with PBS, and resuspended into antibiotic-free cell culture medium. H. pylori was added to AGS cells at a bacterium/cells ratio of 100:1, and cultured for the indicated time periods.

Measurements of cell proliferation

The cell proliferation was determined by Trypan blue exclusion assay. The cells were plated at a density of 1 × 10⁵ cells/well in six-well plates and maintained in RPMI 1640 containing 10% FBS at 37°C under microaerophilic conditions for 3 days. The media was changed with serum-free RPMI and incubated for 24 h to arrest cell growth and silence gene activity, followed by treatment with H. pylori at a bacterium/cells ratio of 100:1 for the indicated time periods. After incubation, the cells were washed twice with HBSS and harvested. The number of viable cells was determined using light microscopy and counting cells that excluded the 0.2% trypan blue dye. Cells were counted in a randomized manner using a hematocytometer.

Urease activity assay

Urease activity was measured to test H. pylori's ability to secrete the urease enzyme, which catalyzes the conversion of urea to ammonia and carbon dioxide. Briefly, autoclaved christensen's urea agar was mixed with urea and a bacterial suspension containing the liquid reaction mixture was added to the tube, followed by incubation for 5 h at 37°C under microaerophilic conditions. The reaction was observed for changes in the color, which would indicate an increase in the pH due to the production of ammonia.

Western blot analysis

AGS cells were allowed to grow for 3 days, and were stimulated as described. Cell lysates were prepared using lysis buffer (20 mM tris-HCl [pH7.4], 0.5 mM EDTA, 0.5 mM EGTA, 10 mg/mL triton X-100, 100 μg/mL SDS, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mM PMSF, and 0.7 μg/mL β-mercaptoethanol). The lysates were subjected to electrophoresis on a 12% SDS-PAGE, transferred to nitrocellulose membranes using the Power Pac 1000 (Bio-Rad, Melville, NY, USA) power supply and examined for expression of IκB-α, IL-8, and IL-1β. Actin was used as a loading control. The results were analyzed by Quantity One analysis software (Bio-Rad Chemical Division, Richmond, CA, USA).

Data analysis

All data are expressed as mean ± SEM of 3–4 experiments. Statistical differences among the groups were analyzed by Student's t-test. Data were considered at P < .05.

Results

Changes in cell growth rate during H. pylori infection in AGS cells

Trypan blue assays were performed in cultured AGS cells to investigate the cytotoxic effects of H. pylori. As shown in Figure 1, serum-starved AGS cells were incubated with H. pylori at a multiplicity of infection of 100 for 0, 3, 6, 12, and 24 h, and the cell proliferation was measured using the trypan blue assay. Apoptotic cells were more apparent in cells incubated with H. pylori than those in the control groups at every time point, with the most significant difference seen at 24 h. Infected cell numbers were gradually increased time-dependently. However, the number of live cells in the H. pylori group was decreased to ∼72% compared with cells in the control group.

FIG. 1.

Effect of Helicobacter pylori on the cell proliferation of AGS cells. AGS cells were incubated with H. pylori (multiplicity of infection [MOI]: 100) for the indicated time periods. Cell proliferation was estimated using Trypan blue assay. Data are expressed as mean ± standard error (SE) of three experiments (Student's t-test; *P < .05 vs. control, **P < .01 vs. control). AGS, adenocarcinoma gastric cell line.

Expressions of IκB-α, IL-8, and IL-1β after H. pylori incubation

To investigate when the proinflammatory proteins induced by H. pylori would reach maximal expression levels in AGS cells, serum-starved AGS cells were exposed to H. pylori for 0, 0.5, 1, 2, 4, 6, 8, and 12 h and IκB-α, IL-8, and IL-1β protein expressions were detected by western blot analysis. H. pylori infection significantly degraded IκB-α, which translated into the activation of NF-κB (Fig. 2a). IκB-α expression levels increased after 60 min. It has been reported that H. pylori infection increases the expression of some proinflammatory cytokines, including IL-8 and IL-1β, in gastric mucosa.¹⁷ After incubation with H. pylori, an increase in IL-8 levels was observed relative to cells incubated with medium as a control. IL-8 levels rose steadily and peaked at 8 h, and slightly declined by 12 h (Fig. 2b). AGS cells incubated with H. pylori for 4 h resulted in time-dependent increase in IL-1β protein. After 4 h, the IL-1β expression level decreased (Fig. 2c). Based on these results, experiments investigating IκB-α in AGS cells were performed with H. pylori infection for 1 h and experiments looking at IL-8 and IL-1β were conducted for 8 and 4 h, respectively.

FIG. 2.

H. pylori-induced (a) IκB-α, (b) IL-8 and (c) IL-1β expressions in AGS cells. Serum-starved AGS cells were cocultured with H. pylori (MOI: 100) for the indicated time periods. Data are expressed as mean ± SE of three experiments (Student's t-test; *P < .05 vs. control, **P < .01 vs. control).

H. pylori-induced IL-1β and IL-8 expressions are decreased by ECQ pretreatment

To examine whether ECQ attenuates IκB-α, IL-8, and IL-1β expressions in AGS cells, the cells were pretreated with ECQ for 12 h at 12.5, 25, 50, and 75 μg/mL and exposed to H. pylori. IκB-α, IL-8, and IL-1β expressions were measured by western blot analysis. There was no significant reduction in H. pylori-induced IκB-α degradation in cells pretreated with ECQ (Fig. 3a). ECQ suppressed IL-8 production by H. pylori in a dose-dependent manner (Fig. 3b). Moreover, IL-1β levels were also significantly downregulated by ECQ suggesting it potently inhibited H. pylori-induced inflammation in AGS gastric epithelial cells (Fig. 3c).

FIG. 3.

Effect of ECQ on (a) IκB-α, (b) IL-8 and (c) IL-1β expressions in H. pylori-infected AGS cells. Serum-starved AGS cells were preincubated in the presence of ECQ for 12 h at the indicated concentration. Cells were then stimulated with H. pylori (MOI: 100) for the indicated time periods. Data are expressed as mean ± SE of three experiments (Student's t-test; *P < .05 vs. control, ^# P < .05 vs. H. pylori alone, ^## P < .01 vs. H. pylori alone).

To further address ECQ's ability to inhibit H. pylori-induced inflammation, we examined the effects of ECQ with various combinations of drugs commonly used to treat H. pylori infection (Fig. 4). ECQ in combination with amoxicillin and clarithromycin reduced cytokine expressions in AGS cells more effectively than the ECQ treatment and amoxicillin plus clarithromycin treatment groups. However, omeprazole treatment was not efficacious in the prevention of H. pylori-induced inflammation.

FIG. 4.

Effect of ECQ, amoxicillin, clarithromycin, and omeprazole on (a) IL-8 and (b) IL-1β expressions in H. pylori-infected AGS cells. Serum-starved AGS cells were preincubated in the presence of ECQ (12.5 μg/mL for IL-1β, 50 μg/mL for IL-8) for 12 h before pretreatment with amoxicillin (1 mM, 1 h), clarithromycin (20 μM, 1 h), or omeprazole (20 μM, 1 h). Cells were then stimulated with H. pylori (MOI: 100) for the indicated time periods. Data are expressed as mean ± SE of three experiments (Student's t-test; *P < .05 vs. control, ^# P < .05 vs. H. pylori alone, ^## P < .01 vs. H. pylori alone).

Urease activity of H. pylori is not altered by treating with ECQ

To investigate whether ECQ inhibits the urease activity of H. pylori, an assay using Christensen's Agar Base with urea was conducted. First, urease activity test was performed with or without yeast extract. Without yeast extract, the urease activity of H. pylori was lower compared with the activity in the presence of yeast (Data not shown). We also examined the urease activity of H. pylori incubated with various concentration of ECQ to determine whether it inhibited the urease activity of H. pylori (Fig. 5). H. pylori was combined with Christensen's Agar Base and ECQ. After 5 h, few changes in the liquid medium's pH due to the production of ammonia by urease detected via phenol red, indicating that ECQ did not inhibit the urease activity of H. pylori.

FIG. 5.

Effect of ECQ on urease activity of H. pylori in AGS cells. Cultured H. pylori was incubated in combination with Christensen's Agar Base with urea and ECQ at the indicated concentration. All the reactions were carried out at 37°C. When the urease in H. pylori increases the pH of the medium, the phenol red causes it to change from yellow to red. Color images available online at www.liebertpub.com/jmf

Discussion

The main findings of this study are that H. pylori mediates the degradation of IκB-α and induces proinflammatory cytokines in AGS gastric epithelial cells. In addition, ECQ could be used as a chemoprotective agent for gastritis or peptic ulcers induced by H. pylori. Inflammation mediated by IL-8 and IL-1β is a central theme in H. pylori-induced gastritis and ulcers. Some reports have demonstrated that IL-1β plays an important role in synthesizing IL-8 in response to the inflammatory signals in gastric epithelial cells.^18,19 IL-1β also upregulates many proinflammatory proteins, including IL-8, in other cells types such as esophageal and colon epithelial cells.^20,21 IL-8 is a chemokine that attracts neutrophils and eosinophils.²² These white blood cells are implicated in gastritis and GI tract inflammation. Therefore, IL-8 might be a potential target for therapeutic intervention in gastric inflammation.

Numerous medicinal products have been used for gastritis and ulcers, regardless of their side effects. Many plant extracts are displaying efficacy in treating gastritis, and have become one of the most promising sources of new drugs.²³ Flavonoids such as QCG have anti-inflammatory, antioxidant, antiallergic, anticarcinogenic, and antiviral activities against various diseases.²⁴

Quercetin has been identified to have anti-inflammatory,²⁵ antithrombotic,²⁶ antitumor,²⁷ and antibacterial²⁸ effects. QGC is a quercetin derivative, which is extracted from Rumex Aquaticus Herba. In our previous study, QGC inhibited gastric acid output, which prevented the development of reflux esophagitis.¹³ QGC also reduced ROS generation and ERK activation induced by ethanol in cultured feline esophageal epithelial cells.²⁹ In addition, QGC prevented indomethacin-induced gastric mucosal injury by increasing mucus secretion and attenuating the release of free radical and proinflammatory cytokines.³⁰

In this study, cell growth inhibition induced by H. pylori increased time-dependently with apoptotic cell death. AGS gastric cell proliferation was slightly decreased in the H. pylori-infected groups compared with the control group over 24 h. These results indicate that H. pylori infection may continuously suppress the growth of AGS gastric epithelial cells.

Previous studies suggested that NF-κB activation might be a necessary precondition for IL-8 upregulation in response to H. pylori. ^31
–33 H. pylori adheres to gastric epithelial cells and activates NF-κB, a known regulator of IL-8 production.^31,34
–36 Activation of NF-κB is directly mediated by the degradation of IκB. In this study, IκB-α degradation was evident within 30 min of H. pylori infection and peaked at 1 h.

Our result shows that cytokine expression gradually increased in H. pylori-infected AGS cells, indicating that H. pylori-induced inflammation may mediate an upregulation of IL-8 and IL-1β through the degradation of IκB-α. In addition, we found that pretreatment with ECQ markedly decreased the expressions of H. pylori-induced cytokines. The initial migration and activation of inflammatory cells is known to rely on various proinflammatory cytokines production, especially IL-8.³⁷ These results indicate that the protective effect of ECQ might be related to anti-inflammatory properties that prevent cytokine formation.

Our results also suggested a possible application of ECQ in combination with commonly used antibiotics. In addition to ECQ, two antibiotics displayed an inhibitory effect against H. pylori-induced inflammation in AGS cells. When combined with clarithromycin and amoxicillin, ECQ demonstrated cumulative activity against H. pylori-induced proinflammatory cytokine expressions to almost basal levels. Our results are supported by in vivo studies that demonstrate the protective effects of ECQ on gastric mucosa in gastritis-induced rats.^38,39 The present results indicate that ECQ might have a useful role as part of a combination treatment with anti-H. pylori drugs in the treatment of diseases caused by the bacteria.

In this study, urease activity was detected in live H. pylori. Urease activity of H. pylori is important for gastric colonization and maintaining the cytoplasmic pH.^40,41 We also co-incubated ECQ and H. pylori in Christensen's Agar Base containing urea. However, H. pylori was able to change the pH in the presence of ECQ, indicating that the inhibitory mechanism of ECQ on H. pylori does not involve suppressing urease activity.

Taken together, our findings demonstrate that ECQ has its ability to inhibit the cellular processes critically involved in the pathogenesis of H. pylori, which induces the expressions of various inflammatory cytokines, such as IL-8 and IL-1β. Among the previously mentioned cytokines, the upregulation of IL-8 plays a pivotal role in the H. pylori-mediated signaling pathway in gastric epithelial cells. Further studies need to be conducted to obtain more detailed information about the molecular mechanism of ECQ, but this study supports its potential as a possible candidate for the treatment of gastric inflammation associated with H. pylori infection.

Footnotes

Acknowledgments

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (no. 2011-0012139).

Author Disclosure Statement

No competing financial interests exist.

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