Role of Pediococcus Acidilactici J9 in Decreasing the Occurrence of Gastritis Caused by H.pylori Infection and Two-week Repeated-dose Oral Toxicity Study in Mice

BACKGROUND Helicobacter pylori (H. pylori) is an important pathogen that causes chronic gastritis and peptic ulcer, and is related to the development of gastric carcinoma. Several chemicals, including antibiotics, have been used to eradicate H.pylori. However, more studies are yet requred to accomplish a sucient therapy. Pediococcus acidilactici J9 were studied for inhibition of binding of H.pylori binding to human gastric cell lines. This study was performed in order to investigate the repeated-dose toxicity of Pediococcus acidilactici J9 in male and female mice. RESULTS C57BL/6 male and female Mus musculus were divided into four groups (n = 10 in each group). Pediococcus acidilactici J9 was administered daily by oral injection of vehicle control at dosage levels to a low-dose group (500 mg/kg/day), middle-dose group (1000 mg/kg/day), and high-dose group (2000 mg/kg/day) for two weeks. After 14 days of exposure, the blood biochemistry and hematology were investigated, along with a histopathology exam. There were no bacterial-related deaths or abnormal clinical signs in either gender of mouse. The data was observed during the period in terms of body weight, food, intake, and water consumption. Also, no alterations in organ weights upon administration of Pediococcus acidilactici J9 alone were observed. CONCLUSIONS These results suggest that the oral application of Pediococcus acidilactici J9, up to a dosage level of 2,000 mg/kg/day, causes no adverse effects in both male and female mice. Pediococcus acidilactici J9 inhibits the adhesion of H.pylori to AGS gastric cancer cells. When used as probiotics, Pediococcus acidilactici J9 may help decrease the occurrence of gastritis and reduce the risk of H.pylori infection with promising safety issues.


Background
Lactobacillus is a gram-positive microorganism that utilizes carbohydrates as the energy source and produces organic acids like lactic acid and acetic acid as nal products. It is used industrially in various fermented products, like fermented vegetables and dairy products, which are broadly involved in the everyday life [1,2]. Lactobacillus determines the avor of fermented foods and the characteristics of fermented products, and it plays a critical role in the food preservation. This occurs by extending its shelf life via production of active antibiotic materials like organic acid and bacteriocin. Moreover, various function in the human body is also reported, such as the suppression of intestinal noxious bacteria, the decrease of blood cholesterol levels, anticancer effect, reinforcement of immune function, etc. Because of the Lactobacillus, as a probiotics, intakes living strain, it attracts attention as an antibacterial preparation that solves the residual tolerance problems, in addition to being recently utilized as a healthy functional food [3,4].
Helicobacter pylori is a macroaerophilic gram-negative bacteria that causes chronic gastritis, peptic ulcer, and presumable gastric cancer. Accumulated evidence demonstrates that the eradication of these bacteria resolves H.pylori-associated disease [5]. Multicenter studies have shown that triple therapy via a proton pump inhibitor (PPI), clarithromycin, and either amoxicillin or metronidazole (all taken twice daily).
This therapy is among the most effective approaches to H. pylori eradication [6]. However, 5-10% of H. pylori strains are reportedly resistant to clarithromycin [7]. In addition, there was a study noted a clarithromycin-resistant mutation in 63% of H. pylori strains from patients in whom treatment with a regimen including clarithromycin was unsuccessful [8]. The treatment of H. pylori infection with antibiotics does not eradicate the organism and is also often accompanied by deleterious side effects [9].
Thus, although many experts believe that "untreatable" H. pylori is just ill-treated H. pylori, no clinical trial. To the best of our knowledge, H. pylori has not yielded a treatment that provides 100% eradication [10]. Recently, probiotic lactic acid bacteria (LAB) have been reported to control H. pylori.. Also, several studies have examined the e cacy of various probiotic preparations for H. pylori eradication with and without cointerventions [11]. Moreover, a number of clinical trials have been undertaken to test the hypothesis that probiotic bacteria inhibits H. pylori infection [12]. Probiotics inhibit enteric pathogens such as Salmonella, Shigella, and Citrobacter rodentium in both in vitro and in vivo [13,14], and potential clinical bene ts in preventing or resolving gastrointestinal diseases have been demonstrated [15,16].
These microorganisms provide gut protection through several mechanisms, including decreasing luminal pH by producing lactic acid [17,18] and competing with gut pathogens for host surface receptors [19].
Nonetheless, it has been shown that probiotics may inhibit H. pylori growth, independent of pH and lactic acid levels [20]. In vitro assays were carried out to determine whether the combination of Pediococcus acidilactici J9, and its adhesion to gastric cells thus impacting gastric acidity, inhibit the growth of H. pylori. The current therapeutic regimen for H. pylori aims to eliminate bacterial growth with antibiotics and this reduces the total acidity of gastric acid.
In this study, repeated toxicity tests are performed as the stability test. using mice of C57BL/6 type under the "standard of toxicity test for medicine and medical supplies (Korea food and drug administration noti cation No. 1999-61)". We also demonstrated in in vitro models that Pediococcus acidilactici J9 in combination have bene cial effects similar to those of antibiotic therapy on H. pylori-infected gastric epithelium.

Results
Death rate and normal symptoms Pediococcus acidilactici J9 was administrated by oral injection for 2 weeks and the Table 1, 2 show the death rate and the normal symptoms of males and females observed for 2 weeks. During the experiment, experimental mice were observed at regular times, and no death was observed in the male and female administration group (Table 1). Also, during all of the experiment, in every administration group -low dose (500 mg/kg/day), medium dose (1,000 mg/kg/day), and high dose (2,000 mg/kg /day) -including the control group, no speci c adverse symptoms are observed (Table 2). In this study, a dose of 2,000 mg/kg, which is a maximum dose of oral administration toxicity test, did not generate abnormal symptoms. It thus seems that the minimal lethal dose of this experimental materials exceeds 2,000 mg/kg/day in both male and female.

Changes In Body Weight
Pediococcus acidilactici J9 was orally administrated for 2 weeks with varying concentrations and the changes in body weight are shown in Table 3. Changes in body weight during the whole period of experiment were negligible for the control group, low dose group (500 mg/kg/day), medium dose group (1,000 mg/kg/day), and high dose group (2,000 mg/kg/day). Additionally, from the date of administration of experimental materials to the end of the experiment, there was a normal weekly increase in body weight in the control group and the administration group (Fig. 1A).

Intake Of Nutrition And Water
There was no signi cant change in the control group and the experimental material administration group in the amount of intake of feed and water during the experiment period (Tables 4 and 5). Therefore, it seems that the administration of experimental materials does not affect signi cantly the amount of intake of feed and water ( Fig. 1B and C).

Autopsy Results
As the result of the observation of main organs with naked eyes after the autopsy of experimental mice, there was no signi cant change in organs and speci c autopsy opinion dependent on the dose of administration (Table 6). However in both control group and administration group, blackish red discoloration at the spleen terminal, shrinkage of the right testicle, and thinning of the right atrium were   The Weight Of Organs The weight of organs were measured after repeated administration of Pediococcus acidilactici J9 which varied to low dose (500 mg/kg/day), medium dose (1,000 mg/kg/day), and high dose (2,000 mg/kg/day) for 2 weeks (Table 7). No changes were observed in the weight of brain, lung, testis, ovary, kidney, heart, spleen, and liver with respect to the administration of experimental materials and no abnormal changes were dependent on dose of administrations. Generally, when the toxic materials were ingested, liver takes the largest effect since the detoxi cation starts at the liver. However, there were no signi cant changes in each group on the observed weight of the liver. From the results above, the administration of Pediococcus acidilactici J9 does not affect the weight of organs. Distribution Width of Retics, CHDWr* after the autopsy using blood auto-analyzer (System SE-9000, TOAMedical Electronics Co., Ltd., Kobe, Japan), no signi cant changes were observed in control and administration groups (p ≤ 0.05) ( Table 8). As a result of hematological examination, both the control group and the administration group were included in normal range and no dependence on dose was observed. This result is similar to the range previously reported in hematological fundamental database of which there is a repeated toxicity test for 2 weeks using mice. Blood biochemical analysis.
As the result of the measurement of Glucose; GLU, Blood Urea Nitrogen; BUN, Creatinine; CREA, Total cholesterol; T-CHOL, Albumin; ALB, Total Bilirubin; T-BIL, Alkaline Phosphatase; ALP, Aspartate Aminotransferase; AST(GOT), Alanine Aminotransferase; ALT(GPT), Triglyceride; TG, Total protein; TP using auto-analyzer (Hitachi-747, Hitachi Medical Co., Tokyo, Japan) from blood serum, which is the indicator of blood biochemistry, no signi cant changes dependent on the administration of experimental materials were observed in the whole administration groups with respect to the control group. Both the control group and Pediococcus acidilactici J9 administration group showed normal parameters (p ≤ 0.05) ( Table 9).

Histopathology Observations
For the histopathology test of Pediococcus acidilactici J9-administrated mice, liver and kidney were stained by hematoxylin and eosin. As the result of the histopathology test, no lesions were observed in the liver, like infection, necrosis, iron pigmentation, and bilirubin pigmentation. The structure of liver cells were also normal ( Fig. 2A). There was no lesions in the kidney, like infection and necrosis, and no changes were observed in kidney cells (Fig. 2B). Therefore, there is no signi cant changes in liver and kidney, and no extraordinary pathologic abnormality dependent on dose of experimental materials were observed in both the control group and administration group as the result of the histopathology test. This opinion seems to correspond with the long-term change of weight as well as the blood biochemical change.

Inhibition of adhesion and growth of H.pylori in gastric epithelial cells in the presence of Pediococcus acidilactici J9
The adhesion and growth of H. pylori were inhibited by a 24 h treatment of H. pylori and Pediococcus acidilactici J9 at a 200 ug / ml concentration on AGS cells, which are gastric cancer cells. Compared to the control group (AGS cell and H.pylori), the number of H. pylori analyzed by FACS signi cantly (p < 0.01) decreased after incubation of AGS cell with Pediococcus acidilactici J9 for 24 hours. Control biological triplicate groups are also analyzed for statistical options. (Fig. 3 and Fig. S1 in Additional le 1).

Discussion
Pediococcus acidilactici J9 was obtained from a bean paste soup prepared with ground fermented soybeans and bene cially affects to human body by improving its intestinal microbial balances. Pediococcus acidilactici J9 has been emerged as a potential probiotic. Pediococcus acidilactici J9 exerts as an antagonism against other enteric pathogens, primarily through the production of lactic acid and secretion of pediocin. Thermo-stable pediocin is an antimicrobial peptide is known to have a strong activity against food bacteria and pathogenic enteric bacteria [21]. For these reasons, Probiotics including Pediococcus acidilactici J9 were used for commercial healthcare products like beverages and foods. Pediocin secreted by Pediococcus acidilactici J9 has a potential to inhibit other pathogenic bacteria.
H.pylori is known to be an important causative agent of peptic ulcer, gastritis, gastric cancer, or mucosa associated lymphoid tissue lymphoma [22]. Various antibiotics have been used for H.pylori eradication [23]. These antimicrobial agents have been pointed out for various problems such as adverse effects, risk of re-infection due to increased pH, appearance of resistant bacteria, and high cost [24]. Recently, H.pylori inhibitory activity of natural products has been reported as a new treatment method of H.pylori. There is growing interest in probiotic lactic acid bacteria, which can play a role in the treatment of H.pylori by directly acting on H.pylori, with minimal clinical side effects of antibiotics [25].
This study investigated the toxicity and anti-H.pylori effect of Pediococcus acidilactici J9. Daily administration of Pediococcus acidilactici J9 in mice for two week showed no abnormal clinical signs in body weight, hematology, food intake and water consumption. In all test groups, no general symptoms and deaths from the test substance were observed. During the entire test period, body weight continuously increased but no signi cant change was observed with the control group. In addition, there were no signi cant differences in the gross observation, long-term weight change, hematology, blood biochemical and histopathologic examination of the organ in all the test substance administration groups, and all of them were within the normal range. As a result of repeated toxicity test for 2 weeks, Pediococcus acidilactici J9 was judged to be a safe and low-toxic substance. Pediococcus acidilactici J9, inhibits the adhesion of H.pylori to AGS gastric cancer cells. Probiotics refers to living microorganisms that are bene cial to the human body when consumed in moderate quantities [26,27]. Most probiotics known to date are lactic acid bacteria [28,29]. Probiotic bacteria such as lactic acid bacteria and bene cial bacteria survive in the stomach acid and bile acid in the body, reach the small intestine, multiply in the intestines and settle [30,31]. It has a bene cial effect on health in the colon, and these probiotics should be non-toxic and non-pathogenic [32,33]. Ingestion of probiotics not only helps maintain health, it also helps to improve various diseases such as infants, irritable bowel syndrome, and in ammatory bowel disease [34].
Based on our in vivo and in vitro results, when used as probiotics, Pediococcus acidilactici J9 may help decrease the occurrence of gastritis and reduce the risk of H.pylori infection with promising safety issues, without side effects.

Conclusions
In conclusion, we reported the toxicity and anti-H.pylori effect of Pediococcus acidilactici J9. Daily administration of Pediococcus acidilactici J9 in mice for two week showed no abnormal clinical signs in body weight, hematology, food intake and water consumption. Also, Pediococcus acidilactici J9, inhibited the adhesion of H.pylori to AGS gastric cancer cells. Based on our in vivo and in vitro results, when used as probiotics, Pediococcus acidilactici J9 may help decreasing the occurrence of gastritis and reducing the risk of H.pylori infection with promising safety issues, without side effects.

Methods
Model organisms and conditions C57BL/6 mice of 4 weeks of age without certain pathogens are purchased at an amount of 20 males and females each. Normal and healthy mice without any weight loss are used in experiment by clinical observation during 7 days of education. Feeds are the following; solid feeds for laboratory animal are freely offered, and drinking water. The ltration-puri ed water is also freely offered to mice.

Con guration Of Test Group And Set Of Dosage Setting
Dosage was set by MFDs standards. Maximum dosage is set to 2,000 mg/kg/day for both male and female, with the geometric ratio of 1/2, low dose group, medium dose group, and high dose group are set at 500, 1000, and 2000 mg per body weight(kg) respectively. The number of mice in each group are set to organs and brain were observed with the naked eye. The brain, kidney, liver, lung, reproductive organ, heart, spleen, and thymus are extracted and weighed.

Blood Biochemical
The hematologic analysis of the serum is performed the same day of the autopsy, which is collected from

Hematology
Mice fasted for 8 hours before the autopsy are slightly anesthetized with CO 2 . Part of the blood from the exsanguination is EDTA-treated and stored in tubes and then analyzed by blood auto-analyzer (System SE-9000, TOAMedical Electronics Co., Ltd., Kobe, Japan

Statistical Analysis
All values shown in the gures are presented as mean ± standard error. Western blot results were analyzed by Student's t-test. A 2-tailed probability value below 0.05 was considered statistically signi cant. Data were analyzed using SPSS version 17.0 (SPSS Inc., USA).

Declarations
Ethics approval and consent to participate All experimental animal procedures performed were approved by the Institutional Animal Care and Use Committee (IACUC, Approval number: 16-0043-C2A1) of Seoul National University Hospital, which was accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International.

Consent for publication
Not applicable. Changes in body weight and intake of C57BL/6 mice which treated Pediococcus acidilactici J9. Dosage is set by the standard of MFDS. Maximum dosage is set to 2,000 mg/kg/day for both male and female, and with the geometric ratio of 1/2, low dose group, medium dose group, and high dose group are set by per body weight (kg) respectively. a For every animal, change of weight is measured just before the administration of test materials once a week at certain time during 2 weeks. b, c Intake of feeds and water is measured and calculated once a week. Feeds; solid feeds for laboratory animal are freely offered, and drinking water; ltration-puri ed water is also freely offered. N = 10 samples per group.

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