Dietary prevention of Helicobacter pylori-associated gastric cancer with kimchi

Abstract

To prove whether dietary intervention can prevent Helicobacter pylori-induced atrophic gastritis and gastric cancer, we developed cancer preventive kimchi (cpKimchi) through special recipe and administered to chronic H. pylori-initiated, high salt diet-promoted, gastric tumorigenesis mice model. H. pylori-infected C57BL/6 mice were administered with cpKimchi mixed in drinking water up to 36 weeks. Gross and pathological gastric lesions were evaluated after 24 and 36 weeks, respectively and explored underlying molecular changes to explain efficacies. Cancer preventive actions of anti-inflammation and anti-mutagenesis were compared between standard recipe kimchi (sKimchi) and special recipe cpKimchi in in vitro H. pylori-infected cell model. The erythematous and nodular changes, mucosal ulcerative and erosive lesions in the stomach were noted at 24th weeks, but cpKimchi administration significantly ameliorated. After 36th weeks, scattered nodular masses, some ulcers, and thin nodular gastric mucosa were noted in H. pylori-infected mice, whereas these gross lesions were significantly attenuated in cpKimchi group. On molecular analysis, significant expressions of COX-2 and IL-6, activated NF-κB and STAT3, increased apoptosis, and marked oxidative stresses were noted in H. pylori-infected group relevant to tumorigenesis, but these were all significantly attenuated in cpKimchi group. cpKimchi extracts imparted significant selective induction of apoptosis only in cancer cells, led to inhibition of H. pylori-induced proliferation, while no cytotoxicity through significant HO-1 induction in non-transformed gastric cells. In conclusion, daily dietary intake of cpKimchi can be an effective way either to rejuvenate H. pylori-atrophic gastritis or to prevent tumorigenesis supported with the concerted actions of anti-oxidative, anti-inflammatory, and anti-mutagenic mechanisms.

Keywords: Helicobacter pylori; anti-inflammation; anti-mutagenesis; cancer preventive kimchi; rejuvenation.

Figure 1. Biological actions of standard Kimchi (sKimchi) and cpKimchi; comparison in in vitro H. pylori cell model

A. Cell survival by MTT assay MTT assay was done in AGS cells (left) and RGM-1 cells (right) under the challenge with 1, 2.5, 5, and 7.5 mg/ml concentration of sKimchi and cpKimchi soluble extracts, respectively. Significant cytotoxicities were noted with kimchi more than 5 mg/ml concentration only in AGS cells, none in RGM-1 cells even after kimchi more than 5 mg/ml B. Western blot of HO-1 after each kimchi extracts C. Western blot for Bax and cleaved caspase-3 D. Wound healing assay in AGS cells Previous study showed cpKimchi afforded selective cytotoxicity in cancer cells, wound healing assay after each kimchi extracts administration was done in AGS cells. Significantly delay in wound healing was noted in group administered with cpKimchi extracts. E. RT-PCR and Western blot for COX-2, IL-8, and TNF-α in the presence of H. pylori infection (MOI = 10). cpKimchi significantly attenuated H. pylori-induced COX-2 and TNF-α expression.

Figure 2. Ameliorating effects of cpKimchi in H. pylori-infected chronic atrophic gastritis (24 weeks after H. pylori infection)

A. Protocol for H. pylori-associated gastritis model, 24 weeks. 90 mice were grouped into four, uninfected normal group (n = 20), H. pylori-infected control group (n = 30), 1.7 g/kg cpKimchi administered group (n = 20), and 5.0 g/kg cpKimchi administered group (n = 20) to document the efficacy of 24 weeks cpKimchi administration in H. pylori-initiated, high salt diet-promoted gastric damages model. B. Gross and pathological morphology and index according to group Administration of high salt diet after H. pylori infection led to accentuation of chronic atrophic gastritis. In detail, 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 fore-stomach-glandular stomach area (box indicated). X100 C. Gross lesion scores according to group Gross lesion scores were significantly attenuated with cpKimchi administration (p < 0.01). D. Pathological scores according to group H. pylori infection for 24 weeks led to chronic atrophic gastritis presenting with loss of parietal cells, inflammatory cells such as monocytes, lymphocytes, and macrophages replacing gastric glands, and erosive mucosal changes. These changes were significantly decreased in group treated with cpKimchi 5 mg/kg (p < 0.05). The scoring system was described in “Materials and Methods”

Figure 3. Molecular mechanisms explaining anti-inflammatory and rejuvenating action of cpKimchi (24 weeks after H. pylori infection)

A. The changes of immunohistochemical stainings of COX-2, F4/80, and NF-κB p65 expression according to group The increased expressions of COX-2 were one of core pathogenic mechanisms after H. pylori infection. The expressions of COX-2 were significantly increased in control group (p < 0.01). Macrophage and monocytes were intensively increased with H. pylori infection. However, macrophage infiltrations were significantly decreased in group treated with cpKimchi (p < 0.01). When performed immunostaining with NF-κB p65, significantly increased expressions were noted in control group, whereas much attenuated expressions were seen in group treated with cpKimchi (p < 0.01). B. Changes of western blot of COX-2, IL-6 and STAT3 activation according to group On western blot analysis of mucosal COX-2 expression, COX-2 was significantly increased after H. pylori infection (p < 0.01), but its expressions were significantly decreased in group treated with cpKimchi (p < 0.05). In searching for other intervening factors, significantly increased IL-6 and STAT3 activation was seen in control group. cpKimchi significantly attenuated these levels of IL-6 and associated STAT3 activation (p < 0.05). C. PGE₂ changes according to group Measuring PGE2 levels were done with ELISA and mucosal PGE₂ levels were significantly decreased in group treated with cpKimchi 5 mg/kg (p < 0.01). D. MDA levels according to group Oxidative stress relevant to NF-κB activation was reflected with the levels of MDA, index of lipid peroxidation. H. pylori infection significantly increased the levels of MDA, but these levels were significantly decreased with cpKimchi administration (p < 0.05). E. Changes of HO-1 and HSP70 according to group As indirect index of oxidative stress, we have measured the expressions of HO-1 and HSP70, respectively and the expressions of HO-1 and HSP70 were significantly increased in group treated with cpKimchi (p < 0.05). F. Gastric mucins according to group The gastric mucin was significantly decreased after H. pylori infection (p < 0.01), but its levels were significantly maintained with cpKimchi administration.

Figure 4. Prevention of H. pylori-induced gastric tumorigenesis with long-term intake of cpKimchi (36 weeks after H. pylori infection)

A. Protocol for H. pylori-associated gastritis model, 36 weeks. In order to document the influence of cpKimchi onto the changes of tumorigenesis, the animal model was further extendedly observed up to 36 weeks B. Changes of body weights according to group Though the weight losses were significantly noted after H. pylori infection at 2 months post-infection (p < 0.05), significant weight changes were noted after 5–6 months, statistically significantly blocking of weight loss in group administered with 5 mg/kg cpKimchi (p < 0.05). C. Gross and pathological pictures and index according to group On gross evaluation of resected stomach, the following findings were obtained; nodular mucosal changes, thinned gastric mucosa, adenomatous polyps, and tumorous lesion with central ulcerations. The criteria for gross lesion score was shown in “Materials and Methods”. The whole gross lesion index was significantly increased in control group, but significantly decreased in group administered with cpKimchi (p < 0.05). Severe chronic atrophic gastritis, gastric ulcer, gastritis cystica profunda, adenoma, and adenocarcinoma were shown in control group. X 100. The mean pathological scores were significantly increased in control group, but significantly decreased in group administered with cpKimchi (p < 0.05). The scoring system was described in Supplementary table 2. D. The gastric cancer occurrence rate according to group Gastric adenoma and cancer occurrence were significantly increased in control group, but significantly decreased in group administered with cpKimchi.

Figure 5. Molecular mechanisms to explain cancer preventive effects of cpKimchi (36 weeks after H. pylori infection)

A. Changes of COX-2 and inflammatory mediators according to group On western blot and RT-PCR analysis of mucosal COX-2 expression, COX-2 was significantly increased after H. pylori infection, but its expressions were significantly decreased in group treated with cpKimchi. RT-PCR for IL-1β, VEGF, IL-6, MMP-2 was shown according to group and cpKimchi significantly decreased these H. pylori-induced inflammatory mediators. B. The immunohistochemical changes of COX-2 expressions and macrophage infiltrations according to group COX-2 and F/80 expressions were significantly increased in H. pylori infected control group. However, chronic 36 weeks intake of cpKimchi in drinking water significantly decreased COX-2 expressions as well as macrophage infiltration (p < 0.01). C. Western blot for p-STAT3 and IκB-α cpKimchi efficiently inhibited STAT3 activation and significantly inactivated IκB-α (p < 0.001). D. TUNEL staining for apoptosis and the expression of Bax In order to document the rejuvenating and restorative action of cpKimchi, TUNEL staining was done and apoptosis index was calculated according to group. Compatible with TUNEL, Bax expressions were significantly increased in Group II, but apparently decreased with cpKimchi administration. E. Western blot for Bax, NQO1, HO-1, and HSP70 according to group cpKimchi administration significantly increased NQO1 and HO-1 than control group II. HSP70 was significantly decreased in Group II, but preserved in Group III and IV.