Helicobacter ISSN 1523-5378 doi: 10.1111/hel.12229

Dietary Intervention of Artemisia and Green Tea Extracts to Rejuvenate Helicobacter pylori-Associated Chronic Atrophic Gastritis and to Prevent Tumorigenesis Migyeong Jeong,* Jong-Min Park,* Young-Min Han,* Napapan Kangwan,* Sang-Oh Kwon,† Bok-Nam Kim,‡ Won-Hee Kim§ and Ki-Baik Hahm*,§ *CHA Cancer Prevention Research Center, CHA Bio Complex, CHA University, Pangyo, Korea, †S&D Research and Development Institute, Osong, Korea, ‡Department of Tourism Food Service Cuisine, Hallym Polytechnic University, Chuncheon, Korea, §Digestive Disease Center, CHA University Bundang Medical Center, Seongnam, Korea

Keywords Helicobacter pylori, Artemisia extract, green tea extract, anti-inflammation, rejuvenation, antimutagenesis. Reprint requests to: Ki Baik Hahm, MD, PhD, CHA Cancer Prevention Research Center, CHA Bio Complex 335 Pangyo-ro, Bundang-ku, Seongnam, Kyunggi-do 463-400, Korea, and Digestive Disease Center, CHA University Bundang Medical Center, Seongnam 463-749, Korea. E-mail: [email protected] Migyeong Jeong and Jong-Min Park contributed equally.

Abstract Object: As nonmicrobial dietary approach is capable of controlling Helicobacter pylori infection, we evaluated the efficacy of long-term dietary administration of Artemisia and/or green tea extracts on H. pylori-initiated, high-salt-promoted chronic atrophic gastritis and gastric tumorigenesis mouse model. Methods: Helicobacter pylori-infected and high-salt-diet-administered C57BL/6 mice were administered with Artemisia extracts (MP group) and/or green tea extracts (GT group) for 36 weeks in addition to the control group (ES group, gastroprotective drug, ecabet sodium 30 mg/kg, diet pellet). Gross and pathological gastric lesions were evaluated after 24 and 36 weeks, respectively, and their underlying molecular changes were measured in gastric homogenates. Detailed mechanisms were further evaluated in in vitro cell models. Results: The erythematous and nodular changes and mucosal ulcerative and erosive lesions were noted in the control group at 24 weeks. MP, GT, MPGT, and ES groups all showed significantly ameliorated pathologic lesion compared to the control group (p < .05). After the 36 weeks, scattered nodular masses with some central ulcers and thin gastric surface were noted in the control stomach, whereas no tumorous lesion and milder atrophic changes were observed in all MP, GT, and MPGT groups except ES group (p < .05). On molecular analysis, increased expressions of COX-2, TNF-a, IL-6, lipid peroxide, and activated STAT3 relevant to H. pylori infection were significantly decreased with MPGT administration (p < .01), whereas HSP70 was significantly increased. PGDH expressions, core tumor suppressor involved in carcinogenesis, were significantly decreased with H. pylori infection (p < .05), but significantly increased in MPGT group (p < .05). Increased mucosal apoptotic index noted in the control group was significantly decreased with MP and/or GT along with significantly preserved gastric gastroprotective mediators (p < .01) such as mucins, HSP27, and HSP70. H. pylori-induced serum TNF-a and NF-jB activations were significantly decreased with MPGT administration (p < .05). Conclusion: Long-term dietary intake of MP and/or GT can be an effective strategy either to rejuvenate H. pylori atrophic gastritis or to suppress tumorigenesis.

Helicobacter pylori (H. pylori) infection is considered as a major risk factor for gastric cancer, class I carcinogen by IARC, but eradication alone did not explain the whole picture of gastric cancer prevention because additional modification with environmental or genetic factors, including smoking, alcohol, diet, hygiene, and

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amelioration of mutagenic gastric inflammatory activities, is required [1–4]. Nonmicrobial dietary approach for H. pylori has been suggested as a possible next-generation strategy to prevent H. pylori-associated gastric cancer because continuous surveillance is possible with the warranty of safety,

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effectiveness, high performance, and low risk of resistance, after which diet-based trials were reported in the literature with more than 1000 publications in PubMed search (searched by key word of dietary and Helicobacter), increasing year by year. However, the most studies were based on in vitro documentations dealing with either bactericidal or bacteriostatic effects or in vitro elucidation of molecular molecules for H. pylori-associated gastritis. A few studies were also available on animal model or clinical study performed regarding dietary intervention of H. pylori infection [5]. Artemisia extracts or green tea extracts had been reported to impose potential anti-inflammatory, antioxidative, and antimutagenic actions, especially targeted to various gastrointestinal diseases including alcoholinduced gastritis, stress-related mucosal damages, inflammatory bowel diseases, nonsteroidal anti-inflammatory drug-induced gastrointestinal damages, and cancers. Goswami et al. [6] demonstrated that artemisinin from Artemisia annua showed remarkably strong activity against H. pylori infection. These extracts were not cytotoxic and exhibited in vivo potentiality to reduce the H. pylori burden in a chronic infection model, leading to conclusion that b-artecyclopropylmether from Artemisia could be a lead candidate for antiH. pylori therapeutics. Besides GI diseases, Artemisia extracts had been tried for circulatory disorders, such as dysmenorrhea, hematuria, hemorrhoids, and inflammation, and are also used to treat chronic conditions, such as cancers, ulcers, and digestive disorders. Green tea (GT) and their extracts have been acknowledged as possible cancer preventive in long-term cohort study [7]. Under the preliminary study that Artemisia extracts (MP) and/or green tea extracts (GT), alone or together, had imposed significant anti-inflammatory or antiangiogenic activities, histone deacetylator inhibition, and cytoprotective actions against H. pylori infection [8,9], we have performed in vitro H. pylori-infected model and animal experiment of H. pylori -initiated, high-salt-diet-promoted atrophic gastritis and gastric tumorigenesis model and drawn rescuing outcomes that MP and/or GT resulted in significant either rejuvenating or cancer-preventive effects, based on data regarding non-antimicrobial and dietary intervention for H. pylori infection.

Materials and Methods Reagents Preparation Preparation of MP, GT, and MPGT. The raw materials for the preparation of the powders (Lot NO. SD-MPGT-

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001) were obtained from S&D Co., Ltd. (Cheongwon, Korea). The powders were prepared as follows: Artemisia capillaris (MP) was extracted using water for 8 hours at 95 °C
5, and Camellia sinensis L. was extracted using water for 4 hours at 80 °C
5. The extracts were filtered and evaporated under vacuum (below 60 °C, GT). The concentrated extracts were added to the ethanol and the precipitates were then dried. MPGT is the combination of Artemisia and green tea powders. Each of the powder was used separately in the test. We dissolved the powders in DMSO for in vitro experiment and in water for in vivo. The extracted drinking water was changed weekly, 75 mg/kg per group, equivalent with usual general Korean intake dose of extracts.

Reagents. All chemical reagents were obtained from Sigma-Aldrich (St. Louis, USA). DMEM, penicillin, and fetal bovine serum were obtained from Gibco BRL (New York, USA). Western blotting detection reagents were obtained from Bio-Rad Laboratories (Berkeley, USA). Primers for PCR were synthesized by Bioneer (Daejeon, Korea). Reverse transcriptase was obtained from Promega (Wisconsin, WI, USA). Antibodies for acetylated H3, cyclooxygenase-2 (COX-2), HDAC3, Bcell lymphoma 2 (Bcl-2), cleaved caspase-3, PARP, pNF-jB p65, p-STAT3, heat-shock protein 27 (HSP27), HSP 70, and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) were all obtained from Cell signaling Technology Inc. (Danvers, USA). Hydrogen–potassium ATPase was purchased from Abcam (Cambridge, UK). Inducible nitric oxide synthase (iNOS) and b-actin were purchased from Santa Cruz Biotechnology (Dallas, USA).

H. pylori Culture H. pylori strain ATCC 43504 (American Type Culture Collection, a cagA+ and vacA s1-m1 type strain) for in vitro model and Sydney strain (SS1, a cagA+, vacA s2-m2 strain) for in vivo model were used in this study. H. pylori were cultured at 37 °C in BBL Trypticase soy (TS) agar plate with 5% sheep blood (TSAII; BD Biosciences, Franklin Lakes, NJ, USA) under microaerophilic conditions (BD GasPaK EZ Gas Generating Systems, BD Biosciences) for 3 days. The bacteria were harvested in clean TS broth, centrifuged at 3000 9 g for 5 minutes, and resuspended in broth at a final concentration of 109 colony-forming units (CFUs)/mL. In all experiments, cultures grown for 48 hours on TS agar plates were used.

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Disk Diffusion Method Growth inhibition was performed by the filter paper disk diffusion method on BBL Trypticase soy (TS) agar plate with 5% sheep blood under microaerophilic conditions at 37 °C. The samples were dissolved in dimethylsulfoxide (DMSO) and evaluated for their anti-Helicobacter activity. All compounds were assayed against H. pylori strain ATCC 43504; the surfaces of the BBL Trypticase soy (TS) agar were inoculated with 100 lL of bacterial suspensions. Blank standard disks (6 mm in diameter) were deposited on the plates and impregnated with 50 lL of different dilutions of test compounds. Following incubation for 2–3 days at 37 °C, the inhibition zone around each disk (average diameter), if any, was recorded. The control disks received 50 lL of DMSO. All tests were performed in triplicate, and the antibacterial activity was expressed as the mean of inhibition diameters (mm) produced by the tested compounds.

acidity, after which each animal was intragastrically inoculated with a suspension of H. pylori containing 109 CFUs/mL with an equal volume (0.1 mL) of clean TS broth using gastric intubation needles. All groups were given injections of H. pylori total four times for a week. One group of 10 mice (uninfected group) was given injections of clean TS broth. The mice were fed a special pellet diet AIN76 containing 7.5% NaCl to collect data on more exacerbated conditions for 4 weeks. Then, H. pylori -positive mice were randomly divided into five groups (n = 50). Pellet diet AIN76 was administrated for 24 weeks and 36 weeks to promote H. pylori-induced carcinogenic process in all infected animals. Experimental groups are shown in Figs 2A and 5A and all animal studies were carried out in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of CHA University CHA Cancer Institute after IRB approval. Stomach tissues were isolated and subjected to further histologic examination, ELISA, Western blotting, and PCR.

H. pylori -Infected Mice Model Animals. Five-week-old female C57BL/6 mice (Charles River, Tokyo, Japan) were fed sterilized commercial pellet diets (Biogenomics, Seoul, Korea) and sterile water ad libitum and housed in an air-conditioned biohazard room at a temperature of 24 °C. After 1 week of adaptation, 20 mg/kg pantoprazole was injected three times to facilitate H. pylori colonization through lowering gastric

RNA extraction and RT-PCR. Total RNA was isolated from stomach tissues and cells using TRIzol reagent (Life Technologies, Milan, Italy), and 1–5 mg of each total RNA was transcribed to cDNA using the M-MLV reverse transcriptase (Promega) system for RT-PCR using oligo-dt primer. PCR Primer pairs are given in Table 1. The PCR mixture contained 2X PCR Mastermix

Table 1 Primers Primer In vitro COX-2 iNOS IL-6 IL-8 TNF-a VEGF HIF-1a PDGF bFGF HO-1 HSP27 HSP60 HSP70 HSP90 GAPDH In vivo COX-2 IL-6 IL-1b GAPDH

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Sense primer, 50 ?30

Antisense primer, 50 ?30

Size, bp

Anneal temp, °C

GAA ATG GCT GCA GAG TTG AA TTT TCC CAG GCA ACC AGA CG CTT CCA GCC AGT TGC CTT CT CAG ACA GTG GCA GGG ATT CA CCC TCA CAC TCA GAT CAT CTT CTC AA CAA TGA TGA AGC CCT GGA GT AAC AAA CAG AAT CTG TCC TC AGG AAG CCA TTC CCG CAG TT TAT GAA GGA AGA TGG ACG GC GAC AGC ATG TCC CAG GAT TT GAC AGC ATG TCC CAG GAT TT CGC CCC GCA GAA ATG CTT CG GAG TTG AGC GGC ATC CCG CC TGC CAA GAT GCC TGA GGA AA GGT GCT GAG TAT GTC GTG GA

TCA TCT AGT CTG GAG TGG GA GTA GCG GGG CTT CAG AAT GG GAG AGC ATT GGA AGT TGG GG TTG GGG ACA CCC TTT AGC AT TCT AAG GTA CTT GGG CAG GTT GAC CTC GAT TTC TTG CGC TTT CGT TT GGT AAT GGA GAC ATT GCC AG CTA ACC TCA CCT GGA CCT CT AAC AGT ATG GCC TTC TGT CC GGT TCT GCT TGT TTC GCT CT GGT TCT GCT TGT TTC GCT CT CAG CCA ACA TCA CAC CTC TC GTC CTA GAT TCA CAC CTG GAG TGC CAA GAT GCC TGA GGA AA TTC AGC TCT GGG ATG ACC TT

356 398 496 179 428 211 275 249 192 198 569 454 584 497 404

58 58 58 56 58 55 58 60 58 60 64 64 62 60 58

CAT CCT GCC AGC TCC ACC GC CCG GAG AGG AGA CTT CAC AG CAG GCT CCG AGA TGA ACA ACA AAA AAT GTA TCC GTT GTG GAT CT

GGG AGG AAG GGC CCT GGT GT TGG TCT TGG TCC TTA GCC AC TGG GGA ACT CTG CAG ACT CAA ACT TCC ACC ACC CTG TTG CTG TA

474 479 332 300

58 58 58 58

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Figure 6 Continued.

changes in week 24, serum levels of TNF-a were significantly increased in 36 weeks of H. pylori infection (Fig. 6B). Also, significantly decreased level of serum TNF-a was noted with GT and MPGT administrations (p < .05, Fig. 6B). H. pylori infection significantly increased macrophage infiltration, but MP and/or GT significantly decreased F4/80 immunostainings (p < .05, Fig. 6C). Next, we have measured the levels of NF-jB according to the groups and found that H. pylori infection was associated with significantly increased activation of NF-jB p65 and that MPGT significantly repressed H. pylori-induced NF-jB activation (p < .05, Fig. 6D). 15-PGDH has been acknowledged as a significant tumor suppressor gene in various cancers including H. pylori-associated gastric carcinogenesis [17,18]. As seen in Fig. 6E–G, H. pylori infection led to significant reduction in 15-PGDH expression. However, MPGT administration led to significant preservation of 15PGDH in spite of H. pylori infection, signifying that MPGT played significant anticancer effects through blocking 15-PGDH decrement. Next, we have focused onto the changes in apoptosis and gastric mucin because decrement in gastric mucin makes vulnerable to H. pylori infection and apoptosis induction [19]. As expected, H. pylori infection led to significantly

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increased apoptotic index (p < .001, Fig. 7A), and significantly decreased levels of gastric mucins, high molecular weight glycoproteins expressed throughout the gastrointestinal tract imposing a key role in mucosal protection and restoration, of which reduction has been regarded as delayed healing after damages as well as gastric carcinogenesis, were noted after H. pylori infection (p < .05, Fig. 7B). However, long-term intake of MP and/or GT significantly attenuated apoptosis (p < .01) and significantly preserved gastric mucin (p < .05). Taken together all these results, long-standing dietary administrations of phytoceuticals, Artemisia extracts, and green tea extracts in the current study led to significant rescuing actions from H. pylori-associated chronic gastritis as well as gastric tumorigenesis, signifying that non-antimicrobial dietary approach can be an alternate way to deem uncertainty of microbial- or therapeutic-based approach (Fig. 7C).

Discussion It becomes clear from our study that long-term dietary intervention, as alternative treatment regimens of eradication therapy particularly for patients with antibioticresistant strains of H. pylori and high-risk patients

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TUNEL assay. To detect apoptosis, stomach tissues were stained with the terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling (TUNEL) method using the DeadEndTM Fluorometric TUNEL System (G3250#; Promega).

Immunohistochemistry. Immunohistochemistry was performed on replicate sections of mouse stomach tissues. Sections fixed in 10% buffered formalin and embedded in paraffin were deparaffinized, rehydrated, and boiled three times in 100 mM Tris-buffered saline (pH 7.6) with 5% urea in an 850-W microwave oven for 5 minutes each. Sections were also incubated with F4/80 antibody in the presence of 1.0% bovine serum albumin and finally incubated for 16 hours at 4 °C. The sections were counterstained with hematoxylin and eosin.

In vitro H. pylori-Infected Cell Model Cell culture and cytotoxicity assay. Rat gastric epithelial cell lines (RGM-1) were obtained from Prof. H. Matsui (Tsukuba Univ., Japan). RGM-1 cells were cultured in DMEM. All mediums were supplemented with 10% fetal bovine serum (Gibco BRL) at 37 °C in 5% CO2. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was purchased from Sigma Chemical Co. (St. Louis, MO).

RNA isolation and RT-PCR. Total RNA was isolated from cells using TRIzol reagent (Life Technologies, Milan, Italy), and 1–5 mg of each total RNA was transcribed to cDNA using the M-MLV reverse transcriptase (Promega, Madison, WI, USA) system for RT-PCR using oligo-dt primer. Primer pairs are described in Table 1. The PCR mixture contained 2X PCR Mastermix (K-2018-1; Bioneer), autocraving water, primer (10pmole/lL), and cDNA in final volume of 20 lL. Reactions started at 95 °C for 5 minutes, amplification for 35 cycles (30 seconds at 95 °C, 30 seconds at the annealing temperature listed in Table 1, and 30 seconds at 72 °C), followed by

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a final 7-min extension at 72 °C. Each PCR product was directly loaded onto 1% agarose gels and stained with Redsafe (Cat. No. 21141; iNtRON Biotechnology, Cheonan, Korea).

Western blot. Cells were washed twice with PBS and then lysed in ice-cold cell lysis buffer (Cell Signaling Technology, Denver, MA, USA) containing 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich). After 30 minutes of incubation, samples were centrifuged at 10,000 9 g for 10 minutes. Supernatants were then collected. Proteins in lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes, which were incubated with primary antibodies, washed, incubated with peroxidaseconjugated secondary antibodies, rewashed, and then visualized using an ECL system (GE Healthcare, Buckinghamshire, UK).

Statistical Analysis Results are expressed as the mean
SD. The data were analyzed by one-way ANOVA, and the statistical significance between groups was determined by Duncan’s multiple range test. Statistical significance was accepted at p < .05.

Results Artemisia and/Or Green Tea Extracts Either Downregulated Inflammatory Mediators or Upregulated Various Cytoprotective Proteins in H. pylori-Infected In vitro Cell Model Helicobacter pylori infection [10 multiplicity of infection (MOI), 24 hours] led to significantly increased expressions of Cox-2, iNOS, il-6, and TNF-a mRNA and COX-2 and iNOS protein expressions in normal rat gastric mucosal cells, RGM-1, but 0.1 mg/mL MP and/or GT extracts significantly attenuated the expression of these

Figure 1 In vitro influences of Artemisia and/or green tea extracts in H. pylori-infected cell models; anti-inflammative, anti-angiogenic, cytoprotective, and anti-H. pylori actions (A) Changes in COX-2, iNOS, IL-6, and TNF-a H. pylori infection led to significant expressions of Cox-2 and iNOS mRNA and their protein expressions, but 0.1 mg/mL of MP and/or GT extracts significantly attenuated these inflammatory mediators (B) Changes in H. pylori-induced angiogenic growth factors H. pylori infection was associated with significant inductions of angiogenic growth factors including IL-8, VEGF, HIF-1a, PDGF, and bFGF, but addition of MP and/or GT significantly decreased mRNA of these angiogenic growth factors (C) HDAC3 changes H. pylori infection led to significantly increased expression of HDAC3, additively showing decreased expression of acetylated H3. However, GT and MPGT showed decreased expression of HDAC3 (D) HSP H. pylori infection is associated with the cancelation of HSP70, and the expressions of HSP70 were significantly increased with GT and MPGT administration (E) HSP and caspase-3, PARP, and Bcl-2 H. pylori infection led to increased expressions of caspase-3 cleavage and PARP, but MP and/or GT inhibited caspase-3 cleavage and PARP (F) Anti-H. pylori influence.

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E

F

Figure 1 Continued.

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Figure 2 The influence of Artemisia and/or green tea extracts on H. pylori-induced chronic atrophic gastritis (24 weeks of H. pylori infection) (A) Experimental scheme (B) Gross morphology of resected stomach according to group H. pylori infection followed with high-salt diet led to some erosions, erythematous gastric mucosa, nodular mucosal changes, and protuberant foci of gastric mucosa at forestomach–glandular stomach area. Gross lesion scores were significantly attenuated with MO and/or GT administration in pellet diet (C) The change in mean body weights after H. pylori infection according to group (D) Pathological changes according to group The changes in chronic atrophic gastritis presenting with loss of parietal cells, inflammatory cells such as monocytes, lymphocytes, and macrophages replacing gastric glands, and erosive mucosal changes were prominent in 24 weeks of H. pylori infection. However, these changes were significantly decreased in group treated with MP and/or GT. Table 2 shows the scoring system for pathological changes. The degree of gastric atrophy was specifically determined with immunohistochemical staining of proton-pump antibody.

inflammatory mediators, respectively (p < .05, Fig. 1A). As H. pylori infection induced angiogenic growth factors including IL-8, VEGF, HIF-1a, PDGF, and bFGF to propagate gastric inflammation as well as promote carcino-

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genesis [11], we measured the changes of these angiogenic growth factors in RGM-1 cells. As seen in Fig. 1B, H. pylori infection induced significant inductions of IL-8, HIF-1a, PDGF, and bFGF (p < .01). How-

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D

Figure 2 Continued.

ever, the addition of MP and/or GT significantly decreased mRNA of these angiogenic growth factors (p < .05, Fig. 1B). As histone deacetylator is associated with these increased expressions of H. pylori-associated inflammatory mediators as well as angiogenic growth factors [12] relevant to inflammation-based carcinogenesis in the stomach, we have measured the changes in HDAC3 and acetylated H3 (Fig. 1C). As noted in Fig. 1C, H. pylori infection led to significantly increased expression of HDAC3, additively showing decreased expression of acetylated H3 (p < .05). However, MP and/or GT showed significantly decreased HDAC3 (p < .05), leaving the possibility Artemisia and/or green tea extract might impose anti-inflammatory actions mediated through HDAC inhibition. We have already published that H. pylori infection is associated with the cancelation of heat-shock protein (HSP70) [13], but the expressions of HSP70 were significantly maintained in MP and/or GT treatment (Fig. 1D,E). As H. pylori infection damaged gastric epithelial cells through robust apoptosis [14], we measured the changes in cleaved cas-

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pase-3, PARP, and Bcl-2. As seen in Fig. 1E, H. pylori infection led to increased expressions of caspase-3 cleavage and PARP, but MP and/or GT inhibited caspase-3 cleavage and PARP. Inversely, Bcl-2 was significantly decreased after H. pylori infection, but MPGT preserved induced expression of Bcl-2. When performing disk hemolysis analysis for anti-H. pylori effect, MP and/or GT all significantly increased anti-H. pylori (p < .01, Fig. 1F). Supported with these in vitro actions, we proceeded to in vivo H. pylori-infected animal model.

Artemisia and/or Green Tea Extracts Ameliorated Chronic Atrophic Gastritis in H. pylori-Infected Mice (24 Weeks) Attenuated chronic atrophic gastritis with 24 weeks of dietary administration of Artemisia and/or green tea extracts. After 24 weeks of H. pylori infection in C57BL/6 mice, whose experimental scheme was shown in Fig. 2A, co-administration of high-salt diet led to attenuation of CAG as

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B

C

D

Figure 3 Changes in serum TNF-a, IL-6, PGE2, and MDA (A) Changes in serum TNF-a according to group (B) Changes in serum IL-6 (C) Changes in serum PGE2 (D) Changes in serum MDA.

seen in Fig. 2B,D. On gross examination, H. pylori infection followed with high-salt diet led to some erosions, erythematous gastric mucosa, nodular mucosal changes, and protuberant foci of gastric mucosa at forestomach–glandular stomach area (Fig. 2B). Overall pathological features seen in the control group were compatible findings of chronic atrophic gastritis (CAG) (Fig. 2D). These changes were quite compatible with our previous publication that our H. pylori-infected, high-salt diet mouse model yielded CAG on 24 weeks [15]. Gross lesion scores were significantly attenuated with MP and/or GT administration in drinking water (p < .05, Fig. 2B). Because H. pylori infection is associated with significant decreases in body weight changes as the time after H. pylori infection has increased, MP administration significantly attenuated H. pylori infection-associated body weight loss (p < .05, Fig. 2C). As seen in Fig. 2D, the characteristic pathology of CAG pre-

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senting with loss of parietal cells, which was specifically scored with immunohistochemical staining of protonpump antibody, inflammatory cells such as monocytes, lymphocytes, and macrophages replacing gastric glands, and erosive mucosal changes, was noted after 24 weeks of H. pylori infection. However, these changes were significantly decreased in the group treated with MP and/or GT (p < .05, Fig. 2D), leading to rejuvenation of H. pylori-induced CAG. We speculated that all of these rescuing actions of MP and/or MPGT were based on rejuvenating actions of H. pylori-induced CAG.

Anti-inflammatory actions of MP and/or GT. The increased levels of serum TNF-a and IL-6 were reported after H. pylori infection [16]. As seen in Fig. 3A,B, the mean serum levels of TNF-a and IL-6 were significantly increased in Group 2, but TNF-a and IL-6 were signifi-

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B

C

Figure 4 The changes in inflammatory mediators, apoptosis, and gastric mucins according to group (A) The changes in Cox-2, Il-6, and Il-1b according to group The expressions of COX-2, IL-6, and IL-1b were significantly increased in the control group, whereas expressions of all these were significantly attenuated with each treatment. (B) TUNEL and apoptotic index (AI) according to group H. pylori infection led to significantly increased apoptotic index (p < .01), but MP and/or GT as well as ES significantly decreased these AIs (C) PAS staining for gastric mucins The gastric mucins were significantly decreased after H. pylori infection, but its levels were significantly maintained with MP and/or GT.

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Figure 5 The influence of Artemisia and/or green tea extracts on H. pylori-induced gastric tumorigenesis (36 weeks of H. pylori infection) (A) Experimental scheme (B) Gross morphology of resected stomach according to group H. pylori-initiated, high-salt-diet-promoted gastric tumorigenesis model has produced significant changes including nodular mucosal changes, thinned gastric mucosa, adenomatous polyps, and tumorous lesion with central ulcerations. (C) The change in mean body weights after H. pylori infection according to group (D) Pathological changes according to group On pathological observation, these gross lesions were severe chronic atrophic gastritis, gastric ulcer, gastritis cystica profunda, adenoma, and some mice developed gastric adenocarcinoma.

cantly decreased in groups 3, 4, and 5. PGE2 levels were measured by ELISA. Serum PGE2 levels were significantly decreased in the group treated with MPGT (p < .1, Fig. 3C). Oxidative stress relevant to inflammation was reflected with the levels of MDA, index of lipid

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peroxidation. H. pylori infection significantly increased the levels of MDA, but these levels were significantly decreased with GT administration (p < .05, Fig. 3D). COX-2, IL-6, and IL-1b activations were well-known changes relevant to H. pylori infection. As seen in Fig. 4A,

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D

Figure 5 Continued.

the expressions of COX-2, IL-6, and IL-1b were significantly increased in the control group, whereas expressions of all those were significantly attenuated with each treatment. Robust apoptosis is also the core mechanism for H. pylori-associated gastric damages. As noted in Fig. 4B, H. pylori infection led to significantly increased apoptotic index (p < .01), but MP and/or GT as well as ES significantly decreased these AIs (p < .01). The gastric mucins were significantly decreased after H. pylori infection (p < .01, Fig. 4C), but its levels were significantly maintained with MP and/or GT administration (p < .01).

Artemisia and/or Green Tea Extracts Prevented Gastric Tumorigenesis in H. pylori-Infected Mice (36 Weeks) Artemisia and/or green tea extracts prevented H. pylori-associated atrophic change and gastric tumorigenesis. To document the preventive effects of MP and/or GT on the changes in H. pylori-initiated, salt-promoted gastric

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tumorigenesis, the animal model was further observed up to 36 weeks (Fig. 5A). As seen in Fig. 5B and D, 36 weeks of H. pylori infection led to significant development of gastric tumors as well as appearance of CAG (p < .001). In detail, H. pylori-initiated, high-salt-dietpromoted gastritis model has produced significant gastric tumorigenesis after 36 weeks featured with nodular mucosal changes, thinned gastric mucosa, adenomatous polyps, and some tumorous lesions with central ulcerations. On pathological observation, gross lesions noted in Fig. 5B were severe CAG, gastric ulcer, gastritis cystica profunda, adenoma, and gastric adenocarcinoma (Fig. 5D). These gross lesions as well as increased pathological scores seen in the control group were all significantly ameliorated in group administered with MP and/or GT (p < .05). Although the weight losses were significantly noted after H. pylori infection around 7 weeks, significant weight changes were noted after 29 weeks, statistically significantly blocking of weight loss in group administered with MP and/or GT (p < .05, Fig. 5C).

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Figure 6 Molecular changes according to group implicated in H. pylori-associated gastric tumorigenesis (A) The changes in COX-2, pSTAT-3, and HSP70 molecular mechanisms responsible for cancer-preventive effects of MO and/or GT COX-2 was significantly increased in Group 2, and STAT3 activation was noted in Group 2. However, in group treated with MP and/or GT, all showed significant repression of COX-2 and STAT3 activations (B) The changes in mean levels of serum TNF-a (C) Immunohistochemical staining of F4/80 according to group H. pylori infection significantly increased macrophage infiltration, but MP and/or GT significantly decreased F4/80 immunostainings (D) Western blot of NF-kB p65 according to group (E–G) The changes in 15-PGDH according to group, (E) Western blot, (F) immunohistochemical staining (G) mean levels of 15-PGDH expression according to group.

Molecular mechanisms responsible for cancer-preventive effects of MP and/or GT. Similar to the molecular changes observed at 24 weeks (Fig. 4), COX-2 was significantly increased in Group 2 (p < .05, Fig. 6A). Significant activation of STAT3 was noted in Group 2, but administra-

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tion of MP and/or GT led to significant reduction in COX-2 and STAT3 activations (p < .05, Fig. 6A). H. pylori infection is also associated with the cancelation of HSP70 [12], which was also significantly observed in Group 2 compared to Group 1 (p < .05). Similar to the

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C

D

Figure 6 Continued.

changes in week 24, serum levels of TNF-a were significantly increased in 36 weeks of H. pylori infection (Fig. 6B). Also, significantly decreased level of serum TNF-a was noted with GT and MPGT administrations (p < .05, Fig. 6B). H. pylori infection significantly increased macrophage infiltration, but MP and/or GT significantly decreased F4/80 immunostainings (p < .05, Fig. 6C). Next, we have measured the levels of NF-jB according to the groups and found that H. pylori infection was associated with significantly increased activation of NF-jB p65 and that MPGT significantly repressed H. pylori-induced NF-jB activation (p < .05, Fig. 6D). 15-PGDH has been acknowledged as a significant tumor suppressor gene in various cancers including H. pylori-associated gastric carcinogenesis [17,18]. As seen in Fig. 6E–G, H. pylori infection led to significant reduction in 15-PGDH expression. However, MPGT administration led to significant preservation of 15PGDH in spite of H. pylori infection, signifying that MPGT played significant anticancer effects through blocking 15-PGDH decrement. Next, we have focused onto the changes in apoptosis and gastric mucin because decrement in gastric mucin makes vulnerable to H. pylori infection and apoptosis induction [19]. As expected, H. pylori infection led to significantly

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increased apoptotic index (p < .001, Fig. 7A), and significantly decreased levels of gastric mucins, high molecular weight glycoproteins expressed throughout the gastrointestinal tract imposing a key role in mucosal protection and restoration, of which reduction has been regarded as delayed healing after damages as well as gastric carcinogenesis, were noted after H. pylori infection (p < .05, Fig. 7B). However, long-term intake of MP and/or GT significantly attenuated apoptosis (p < .01) and significantly preserved gastric mucin (p < .05). Taken together all these results, long-standing dietary administrations of phytoceuticals, Artemisia extracts, and green tea extracts in the current study led to significant rescuing actions from H. pylori-associated chronic gastritis as well as gastric tumorigenesis, signifying that non-antimicrobial dietary approach can be an alternate way to deem uncertainty of microbial- or therapeutic-based approach (Fig. 7C).

Discussion It becomes clear from our study that long-term dietary intervention, as alternative treatment regimens of eradication therapy particularly for patients with antibioticresistant strains of H. pylori and high-risk patients

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E

G

F

Figure 6 Continued.

with H. pylori-associated CAG, was very effective in either rejuvenating atrophic gastritis or preventing gastric tumorigenesis. We have shown that dietary administration of phytoceutical extracts, MP and/or GT in this study, significantly imposed anti-inflammatory, antioxidative, antimutagenic, and cytoprotective actions; all of these beneficiary mechanisms orchestrated to prevent inflammation-based gastric tumorigenesis. Even though there were antimicrobial actions of MP and/or GT against H. pylori infection, majorly contributing actions of MP and/or GT were through antimutagenesis based on their anti-inflammation and antioxidation. Not studied in the current experiments, we inferred that after 1 week of triple therapy with PPI, clarithromycin, and amoxicillin, supplemented administration of our phytoceuticals can enhance the eradication rates. In the era of uncertainty about exact eradication guideline except peptic ulcer disease and gastric MALToma, we convinced that long-standing administration of natural products surely mitigated

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H. pylori infection-associated mutagenic actions very efficiently and very safely, but necessitating further clinical study to prove our hypothesis. Remarkably, as core anticancer mechanism in addition to rejuvenating action, because Ryu et al. [15] published that among four H. pylori-infected subjects, the suppression of 15PGDH expression was reversed by H. pylori eradication therapy and these suppressions of 15-PGDH expression were associated with the attenuation of mechanisms involved in H. pylori-associated gastric carcinogenesis including TLR-4 and MyD88 expressions, phosphoERK1/2, and EGF receptor (EGFR)-Snail, our study might be the first to report that dietary intervention could specifically hinder H. pylori-induced 15-PGDH suppression [20]. As for the implication of eradication therapy to achieve cancer prevention, still debates exist even though Japanese strategy that eradication is extendedly indicated for symptomatic chronic gastritis patients in Japan from 2013 is ongoing [21]. Moreover, as increas-

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ing resistance to antibiotics has emerged as standing problem, searches for effective and safer regimen continue. For instance, nonbismuth quadruple (concomitant) therapy appears to be an effective, safe, and well-tolerated alternative to triple therapy and is less complex than sequential therapy [22]. Therefore, so many candidates had been tried as agents for nonmicrobial dietary strategy, and most of the trials were executed in vitro. However, some were clinical trials, not the evidence-based medicine level and cross-sectional results. There had been scant report clarifying the beneficial effects of long-term dietary intervention focused on the rejuvenating efficacy of precancerous lesion or cancer prevention [15,23]. The leaves and stems of Artemisia for a long time have been known to inhibit inflammatory cytokine production and allergic reactions [24]. For instance, Ju et al. [25] found that caspase-mediated activation of the mitochondrial death pathway plays a critical role in Artemisia extract-induced apoptosis of HeLa cells and inhibited the in vivo tumor growth of HeLa xenograft mice; Kim et al. [26] indicated that Artemisia extracts are a potential anti-endometriotic agent, acting to induce apoptosis of endometrial cells through the modulation of the p38 and NF-jB pathways; and Han et al. [27] showed that Artemisia extracts imposed antifibrotic properties via both upregulating antioxidant activities and downregulating the production of extracellular matrix protein in the rat bile duct ligation model. In the current study, we have administered Artemisia extract adjusted onto the amounts ingested as dietary consumption, but for longer periods. Green tea is a very popular beverage and its consumption is associated with lowered risk of several cancers, including stomach, esophagus, pancreas, liver, prostate, lung, and colon cancers. These cancer-preventive effects of green tea have been attributed to its major phytopolyphenols [28], especially more effective in precancerous stage. Among green tea components, (-)-epigallocatechin-3-gallate (EGCG) possesses remarkable cancer-preventive and therapeutic potential against various cancer sites in animal tumor and in some human epidemiologic studies [29,30]. Studies have shown that EGCG possessed diverse pharmacological properties that include anti-

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oxidative, anti-inflammatory, anticarcinogenic, antiarteriosclerotic, and antibacterial effects [31]. In the GI tract, green tea or Artemisia extract was found to activate intracellular antioxidants, inhibit procarcinogen formation, and suppress angiogenesis and cancer cell proliferation. The redox chemistry of these phytoceuticals is responsible for maintaining a balance between cell proliferation and death, and the regulation of signaling pathways such as NF-jB, activator protein1 (AP-1), or MAPK. These phytopolyphenols exert their effects on these pathways separately or sequentially. By modulating critical cell signaling pathways of mutagenesis, they activate cell death signals, induce apoptosis in precancerous or malignant cells, and finally, led to the inhibition of cancer development or progression [32] because polyphenols or other constituents have been demonstrated to act on multiple key elements in signal transduction pathways related to cellular proliferation, differentiation, apoptosis, inflammation, angiogenesis, and metastasis, although complete molecular mechanisms of action are not completely characterized and many features remain to be elucidated [33]. Although still there remain some points to be fortified, the effect of tea consumption or Artemisia intake on human cancers remains inconclusive [34]. Although 14 cohort studies and several case–control studies have been performed on the relationship between tea drinking and stomach cancer since 1966, the results from cohort studies have been disappointing [35–41]. Of the eight cohort studies on green tea, two studies indicated a reduced risk in stomach cancer. Of the six studies on black tea, one study showed increased risk of stomach cancer. The other studies showed no association between tea consumption and stomach cancer risk. No long-term results were available regarding Artemisia intake. This is why we have tried Artemisia or green tea extract and both and more specifically targeted H. pylori-associated gastric carcinogenesis maximizing biological mechanisms. As seen in the current results, dietary intake of MP and/or GT was proved to be very beneficial in halting the progression of H. pylori-associated gastric pathologies: one was rejuvenation of CAG at 24 weeks and the other was prevention of ensuing gastric tumorigenesis at 36 weeks. However, detailed

Figure 7 (A and B) The change in TUNEL and gastric mucin H. pylori infection led to significantly increased apoptotic index (AI) (p < .001), whereas H. pylori infection was associated with significant decrement of gastric mucins (p < .05). However, chronic intake of MP and/or GT significantly attenuated apoptosis and significantly preserved gastric mucin. (C) Schematic summary showing that dietary intake of Artemisia or/and green tea extracts rescues from H. pylori-induced atrophic change and gastric tumorigenesis Long-standing dietary administrations of phytoceuticals, Artemisia extracts, and green tea extracts in the current study led to significant rescuing actions from H. pylori-associated chronic gastritis and gastric tumorigenesis, signifying that non-antimicrobial dietary approach can be an alternate way to deem uncertainty of microbial- or therapeutic-based approach.

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A

B

C

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clinical trials should be followed to set as clinical recommendation. 15

Acknowledgements and Disclosures This research was supported by the grants from Korean Heath Industry Development Institute (KHIDI) and also by National Center of Efficacy Evaluation for the Development of Health Products Targeting Digestive Disorders (NCEED) and supported by grants from the Globalization of Korean Foods R&D program, funded by the Ministry of Food, Agriculture, Forestry and Fisheries, Republic of Korea. We thank Dr. Eun-Hee Kim for technical assistance. Competing interests: None declared.

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