ISSN 1738-6055 (Print) ISSN 2233-7660 (Online) Lab Anim Res 2018: 34(4), 216-222 https://doi.org/10.5625/lar.2018.34.4.216 In vitro and in vivo inhibition of Helicobacter pylori by Lactobacilllus paracasei HP7 Seong-Soo Hong1, Hyun-A Lee2, Joo Yun Kim3, Ji-Woong Jeong3, Jae-Jung Shim3, Jung Lyoul Lee3, Jae-Hun Sim3, Yungho Chung4, Okjin Kim2,* 1Division of Gastroenterology, Vievisnamuh Hospital, Seoul, Korea 2Center for Animal Resource Development, Wonkwang University, Iksan, Korea 3R & BD Center, Korea Yakult Co., Ltd., Yongin, Korea 4Department of Companion Animal and Animal Resources Science, Joongbu University, Geumsan-gun, Korea The efficacy of standard therapeutic strategies for Helicobacter pylori (H. pylori) infection is decreasing over time due to the emergence of drug-resistant strains. As an alternative, the present study investigated the capacity of Lactobacilllus paracasei (L. paracasei) HP7, isolated from kimchi, to inhibit H. pylori growth. The effects of L. paracasei HP7 on H. pylori adhesion and H. pylori-induced inflammation were examined in AGS human gastric adenocarcinoma epithelial cells and a mouse model of H. pylori SS1 infection. L. paracasei HP7 reduced H. pylori adhesion to AGS cells and suppressed the inflammatory response in infected cells by downregulating interleukin-8. H. pylori colonization in the stomach of C57BL/6 mice was demonstrated by rapid urease test, and results showed significant decrease in mice post-treated with L. paracasei HP7. Additionally, L. paracasei HP7 decreased gastric inflammation and epithelial lesions in the stomach of H. pylori-infected mice. These results demonstrate that L. paracasei HP7 treatment can inhibit H. pylori growth and is thus a promising treatment for patients with gastric symptoms such as gastritis that are caused by H. pylori infection. Keywords: Lactobacilllus paracasei, HP7, Helicobacter pylori, AGS cells, Kimchi Received 24 November 2018; Revised version received 2 December 2018; Accepted 3 December 2018 Helicobacter pylori is a Gram-negative, spiral-shaped increase in the prevalence of H. pylori strains resistant to bacterium in stomach that is the major pathogen of metronidazole and clarithromycin [10-12]. Furthermore, chronic gastric inflammation [1] and stomach ulcers [2] some patients reveal allergic side effects to antibiotics, and is related to increased risk of stomach cancer [3,4]. which can occasionally cause adverse effects while Removing H. pylori in the stomach by inoculating failing to treat H. pylori [13]. Long-term inoculation of antibiotis can reduce H. pylori-related gastrointestinal antibiotics to prevent H. pylori infection cannot be diseases [5,6] and alleviate the risk of stomach cancers recommended. It is therefore important to develop new [7]. The standard recommended treatment for H. pylori non-antimicrobial agents to treat H. pylori [14]. therapy is triple combination therapy with two antibiotics- Lactic acid bacteria (Lactobacillus spp.) have been usually clarithromycin with amoxicillin or metronidazole- recommended as an additive agent in the standard and a proton pump inhibitor, which reveals a successful recommended treatment for H. pylori therapy and can eradication result in the beginning [8,9]. However, the improve patient compliance by decreasing antimicrobial efficacy of this triple therapy has decreased over time; agents-associated side effects [15,16]. Lactobacillus the recent therapy rate of <80% is mainly due to an salivarius was reported to inhibit H. pylori colonization *Corresponding author: Okjin Kim, Center for Animal Resource Development, Wonkwang University, 460 Iksandaero, Iksan, Jeonbuk 54538, Korea Tel: +82-63-850-6668; Fax: +82-63-850-7308; E-mail: [email protected] This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 216 Inhibition of H. pylori by L. paracasei HP7 217 in a mice experiment as evidenced by a decrease in H. at 1×106 cells/mL in Ham’s F-12 medium (Sigma- pylori-specific IgG concentrations, while negative control Aldrich) supplemented with 10% FBS and 1% mice were infected by H. pylori and revealed gastritis antibiotic-antimycotic solution and cultured at 37oC in a lesions [17]. In another study, intragastic treatment of a humidified atmosphere of 95% air/5% CO2 (v/v) for culture supernatant of Lactobacillus acidophilus revealed 16 h. When the cells reached 90% confluence, the to inhibit Helicobacter felis infection [18,19]. Additionally, medium was replaced with serum- and antibiotic-free F- L. acidophilus culture supernatant had a partial but long- 12 medium. An overnight culture of H. pylori SS1 and term inhibiting effect on H. pylori infection in humans L. paracasei HP7 was washed twice in sterile PBS and [20]. resuspended in Ham’s F-12 medium. For co-culture of In the present study, we are aimed to study that the bacteria and gastric epithelial cells, H. pylori SS1 cells lactic acid bacterium Lactobacillus paracasei HP7 (1×107 CFU/mL) were added to wells containing 1×106 isolated from kimchi, a fermented vegetable dish widely AGS cells at a cell ratio of 10:1 and incubated for 4 h in consumed in Korea, has inhibitory effects against H. the absence or presence of L. paracasei HP7. pylori in vitro and in vivo. RNA preparation and real-time (RT)-PCR Materials and Methods Total cellular RNA was extracted using TRIzol reagent (Sigma-Aldrich), and 2 µg were reverse-transcribed Bacterial strains and culture conditions using murine leukemia virus reverse transcriptase, 1 mM L. paracasei HP7 was cultured at 35oC for 24 h in dNTP, and 0.5 µg/µL oligo (dT12-18). The cDNA was Man-Rogosa-Sharpe broth (Difco Laboratories, Detroit, used as a template for RT-PCR to detect H. pylori 16S MI, USA) composed of 0.2% dipotassium hydrogen RNA as a measure of the H. pylori infection rate. The phosphate, 0.5% sodium acetate, 0.8% meat extract, reaction was carried out on a QuantStudio 6 Real-Time 0.1% Tween 80, 0.4% yeast extract, 2% D(þ)-glucose, PCR system (Applied Biosystems, Foster City, CA, 0.02% magnesium sulfate, 1% peptone from casein, USA) using SYBR Premix Ex Taq (Takara Bio, Otsu, 0.2% diammonium hydrogen citrate, and 0.004% Japan), with glyceraldehyde 3-phosphate serving as an manganese sulfate. H. pylori strain SS1 (B0890; Korean internal standard. Relative mRNA levels at each time Collection for Type Cultures, Jeongeup, Korea) was point were compared with those in H. pylori-infected cultured overnight at 37oC under microaerophilic control AGS cells. Forward and reverse sequences of conditions in brain-heart infusion broth containing 10% primers for amplifying the H. pylori 16S RNA gene fetal bovine serum (FBS) and was allowed to grow to a were as follows: 5'-TCG GAA TCA CTG GGC GTA A- density of ~2.0×109 CFU/mL. The cultured bacteria 3' and 5'-TTC TAT GGT TAA GCC ATA GGA TTT were then transferred to phosphate-buffered saline (PBS) CAC-3' [21]. before they were used to infect cells. Measurement of IL-8 levels Cell culture IL-8 released by AGS cells infected with H. pylori was AGS human gastric adenocarcinoma epithelial cells detected by enzyme-linked immunosorbent assay (ELISA). (CRL-1739; American Type Culture Collection, Manassas, AGS cells (2×104 cells/well) were seeded in 96-well VA, USA) were cultured in Roswell Park Memorial plates; L. paracasei HP7 cells were added to the cell Institute 1640 medium (Sigma-Aldrich, St. Louis, MO, culture medium 30 min before H. pylori infection for 24 USA) supplemented with 10% heat-inactivated FBS h. AGS cells cultured in the absence of L. paracasei HP7 (Invitrogen, Carlsbad, CA, USA). Antibiotic-antimycotic cells served as a control. The culture supernatant was (Gibco, Grand Island, NY, USA) was added if needed. collected and IL-8 levels were measured with a For analysis of H. pylori-induced interleukin (IL)-8 sandwich ELISA kit (R&D Systems, Minneapolis, MN, production, antibiotics were not added to the culture USA), according to the manufacturer’s instructions. medium. Each sample was tested in triplicate. Inhibition of H. pylori adhesion to AGS cells Animals AGS cells were seeded in 6-well tissue culture plates Specific pathogen-free (SPF) male C57BL/6 mice Lab Anim Res | December, 2018 | Vol. 34, No. 4 218 Seong-Soo Hong et al. weighing 20-24 g were purchased from Samtako Co. Blood analysis (Osan, Korea) and were maintained at the inspection Blood samples were collected from the heart of facility of Wonkwang University (Iksan, Korea) for 1 sacrificed animals and centrifuged at 1000×g for 15 min week before experiments. Thereafter, the mice were at 4oC; the plasma was stored at 80oC until analysis. maintained in an SPF barrier room with regulated Serum titers of anti-H. pylori antibody were measured temperature (23oC±1oC) and humidity (50±5%) on a using the mouse anti-H. pylori antibody (IgG-1) ELISA 12:12-h light/dark cycle. The animals were fed a kit (Cusabio Biotech, Wuhan, China) according to the sterilized pellet diet (2 Mrad radiation) (Purina, Seoul, manufacturer’s instructions. Korea) and sterilized water ad libitum. All studies were performed in accordance with the Guide for Animal Statistical analysis Experimentation of Wonkwang University and were Values for all parameters under study were recorded approved by the Institutional Animal Care and Use for each experimental unit, and statistical analysis was Committee of Wonkwang University (approval no. performed using a general linear model. Values are WKU 16-44). reported as mean±standard deviation where appropriate. The Student’s t test was used for pairwise comparisons. Bacterial inoculation The incidence with 95% confidence interval was H. pylori SS1 was incubated in brain-heart infusion calculated with MiniTab (State College, PA, USA) broth containing 10% FBS overnight at 37oC under a statistical software package.