경희대학교 약학대학과 (주)바이오리듬의 공동연구 결과-(바이오리듬 김치유산균의 항알러지, 항아토피, 항비염에 탁월한 효능 검증)

[변비+아토피치료 장건강 강력 유산균]

메인사이트 : http://biorhythm.kr

생쥐에서 소양(가려움)행동과 수동형 피부과민 반응에 대한 유산균 L. plantarum-K-1 (LP)의 억제효과 (유산균으로 해결)

장세은¹, 트룽헨², 정용현³, 한명주¹, 김동현² (경희대학교 약학대학 미생물.면역학 교실 교수)

: 산학 공동연구 결과물

*1. 경희대학교 식품영양학과, 2. 경희대학교 약학과, 3.(주)바이오리듬

3: 363-883 충북 청원군 오창읍 양청리 686-4 (재)충북테크노파크 보건의료산업센터 101호

   이젠 끝!

알레르기질환, 가려움증,

아토피성피부염, 비염

한국최초 효능보장제 실시

ABSTRACT

AP-1 및 NF-ΚB 활성화를 억제하는 유산균 L. plantarum-K-1 (LP)을 김치에서 분리하여, 생쥐에서 소양(가려움)행동과 수동형 피부과민 반응에 대한 억제효과를 조사 했다.  LP는 phorbol 12'-myristate 13'-acetate (PMA)로 자극한 RBL-2H3 세포(rat basophilic leukemia)에서 전사인자인 NF-κB 및 c-jun 활성화뿐만 아니라 TNF-α 와 IL-4 의 발현을 유의성 있게 억제했다. 

또한 LP는 생쥐에 IgE-antigen 으로 유도된 수동형 피부과민 반응에 대해 강력한 억제효과를 보였으며, 생쥐당 1×10¹⁰ 섭취를 시켰을 때, 87.5%까지 억제하였다.  P는 생쥐당 1×10¹⁰ CFU의 LP을 섭취시켰을 때도 histamine으로 유도한 소양행동을 58.9%까지 억제했다. 

LP는 히스타민에 의해 야기된 혈관 삼투압성을 크게 저해시켰다. LP는 histamine으로 유도한 혈관삼투압 증가를 억제하였으며, 이 효과는 LP의 소양행동 억제효과와 정비례하였다.  LP ( 1×10¹⁰ CFU)를 histamine의해 유도된 IL-4, IL-1β, TNF-α의 발현을 각각 88.9%, 88.6%, 98.9%을 저해했다. LP는 histamine에 의해 유도된 IgE도 85.3%까지 억제시켰다.

결과적으로 LP는 면역세포에서 전사인자인 NF κB 및 AP-1의 활성화를 억제하여 IgE-switching cytokine IL-4 및 proinflammatory cytokines IL- 1β와 TNF-α 발현을 조절하여 항알러지 효과를 나타내며, 알레르기 질환, 아토피성 피부염, 비염,  가려움증  등의 질병을 개선 할 수 있다.

 * 실제 (주)바이오리듬 유산균 L-plantarum K-1을 2주 동안 섭취후 완전 치료상태로 변한 모습 - 2012년 5월 7일~19일<2주 섭취>,  성인 31세)

LEGENDS

Fig. 1. Lactobacillus plantarum K-1의 PMA로 자극한 RBL-2H3세포의 IL-4 (A), TNF-α (B)의 발현 및 그 전사인자 AP-1 과 NF-κB 의 활성화 억제효과( C). RBL-2H3 세포(2 × 10⁵)는 20 nM PMA (PM)로 자극하고, 불활성화시킨 L. plantarum K-1 (LPL, 0.2 × 10⁹CFU/well LPH, 1 × 10⁹ CFU/well)을 처리했다. Normal group (NOR)은 L. plantarum K-1 대신 부형제만 처리 했다. TNF-α와 IL 4 ELISA와 NF-κB와 AP-1 등은 immunoblotting에 의해 측정했다. Mean ± SD (n=5). # p < 0.05 vs. normal control group. * p < 0.05 vs. PMA-treated group.

Fig. 2. 생쥐에 IgE로 유발시킨 수동형 피부과민 반응에 Lactobacillus plantarum K-1의 억제효과. 수동형 피부 과민 반응은 생쥐의 등에 항 DNP-HSA의 내피 주사로 유도했다.  K-1 및 azelastine에는 3 일 동안 하루에 한 번 경구투여하였다. 등 피부 (1 × 1 ㎝)에서 삼출된 Evan blue 량으로 측정 했다.  NOR, normal group 은 부형제만을 투여; CON, IgE-antigen 만을 투여; LPL, IgE-antigen과 L. plantarum K-1 (1 × 10⁹ CFU) 투여; LPH, IgE-antigen과 L. plantarum K-1 (1 × 10¹⁰ CFU) 투여; AZ, IgE-antigen과 10 mg/kg azelastine 투여. Mean ± SD (n=5). # p < 0.05 vs. normal control group. *

 Fig. 3. 생쥐에 히스타민 유발 소양행동에 Lactobacillus plantarum K-1의 효과.

(A) 소양행동 억제효과. 정상군(Normal control group)은 saline만 투여하고, 실험군은 히스타민을 등에 주사하고, 소양행동을 1시간 측정했다. IgE-antigen만을 처리한 group. LPL은 histamine과 LP (1 × 10 ⁹ CFU/mouse) 투여하고, LPH는 histamine 과 LP (1 × 10¹⁰ CFU/mouse)를, AZ는 histamine과 azelastine (10mg/kg)을 하루에 한번씩 3 일 동안 투여했다. NOR, normal group ; CON,  histamine 만 투여; LPL, histamine과 L. plantarum K-1의 1×10⁹ CFU 투여; LPH, histaminer과 L. plantarum K-1의 1×10¹⁰ CFU 투여; AZ10, 히스타민과 10 mg/kg azelastine 투여                                           

(B) 혈관 삼투압 효과. 생쥐에서 히스타민에 의해 증가됐다. 생쥐의 등에 히스타민 300 μg/50 μL의 주사 1시간 전에 마지막 유산균을 경구투여했다. 혈관투과성시험에 있어서 생쥐의 등표피(1×1 cm)로부터 Evan blue 내출혈의 량이 측정됐다. Mean ± SD (n=5). # p < 0.05 vs. normal control group.*p < 0.05 versus histamine-treated group

Fig. 4. 히스타민으로 유도한 생쥐의 피부조직에서 TNF-α (A) and IL-4 (B), IL-1β (C) 및 IgE (D)의 발현에 Lactobacillus plantarum K-1의 저해효과. TNF-α (a), IL-4 (b), IL-1β (c) 및 IgE (d)는 ELISA.로 측정했다. L. plantarum K-1과 azelastine은 3일동안 하루에 1번 쥐에 경구투여 됐다. NOR, normal group; CON, histamine 만을 투여; LPL, histamine 과 L. plantarum K-1(1x10⁹ CFU ) 투여; LPH, histamine과 L. plantarum K-1(1x10¹⁰ CFU) 투여; AZ, histamine 과 10 mg/kg azelastine 투여. Mean ± SD (n=5). # p < 0.05 vs. normal control group. * p < 0.05 vs. histamine-treated group.

Fig. 5. 히스타민으로 유도한 생쥐 피부의 NF-κB 와 c-jun 활성화에 Lactobacillus plantarum K-1 의 억제효과. NF-κB, c-jun, β-actin은 immunoblot 분석에 의해 측정했다. L. plantarum K-1과 azelastine은 3일동안 하루에 1번씩 쥐에 경구투여 했다. NOR, normal group; CON, histamine만을 투여; LPL, histamine 과 L. plantarum K-1(1x10⁹ CFU ) 투여; LPH,  histamine 과 L. plantarum K-1(1x10¹⁰ CFU) 투여; AZ, histamine과 10 mg/kg azelastine 투여.

(주) 바이오리듬

www.biorhythm.kr  TEL : 010-5509-3307  Email : tenstens@hanmail.net

원문 : Inhibitory Effect of Lactobacillus plantarum K-1 on Passive Cutaneous Anaphylaxis Reaction and Scratching Behavior in Mice

Se-Eun Jang1, Hien-Trung Trinh2, Yong-Hyun Chung3, Myung Joo Han1 and Dong-Hyun Kim2,* 1Department of Food and Nutrion, and 2Department of Life and Pharmaceutical Sciences, Kyung Hee University, 1, Hoegi, Dongdaemun-ku, Seoul, 130-701, Korea 3Biorhythm Co., 685-1, Yangcheongri, Ochang-gun, Choongchungbook-do, 363-883, Korea Running title:

Antiallergic effect of Lactobacillus plantarum K-1 *Correspondence Prof. Dr. Dong-Hyun Kim, College of Pharmacy, Kyung-Hee University, 1, Hoegi, Dongdaemun-ku, Seoul 130-701, Korea E-mail: dhkim@khu.ac.kr Fax: +82-2-957-5030

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ABSTRACT Lactobacillus plantarum K-1 (LP) inhibiting AP-1 and NF-κB activation was isolated from kimchi, and its inhibitory activity against scratching behavior and the passive cutaneous anaphylaxis reaction in mice was investigated. LP potently inhibited the expression‎ of TNF-α and IL-4 as well as the activation of their transcription factors, NF-κB and c-jun, in phorbol 12'-myristate 13'-acetate (PMA)-stimulated RBL-2H3 cells. LP showed potent inhibition against the passive cutaneous anaphylaxis reaction induced by the IgE-antigen complex in mice, inhibiting it by 87.5% at a dose of 1×1010 CFU per mice. LP also potently inhibited histamine-induced scratching behavior by 58.9% compared to the control group at a dose of 1×1010 CFU/mouse. LP significantly inhibited vascular permeability induced by histamine. The inhibitory activity of LP against vascular permeability was in proportion to its inhibition against scratching behavior. LP potently inhibited histamine-induced cytokine production: at 1×1010

Allergic diseases was first proposed to be less preval‎ent in children who were exposed frequently to infectious agents in 1989 (Strachan, 1989). This proposal may be strongly related to the increasing preval‎ence of immune disorder in industrialized countries, which has been attributed CFU per mice it inhibited IL-4, IL-1β and TNF-α expression‎ by 88.9%, 88.6% and 98.9 %, respectively. LP also inhibited IgE levels increased by histamine by 85.3%. It inhibited histamine-induced activation of the transcription factors, NF-κB and AP-1. Based on those findings, LP may improve allergic diseases, such as anaphylaxis, atopic dermatitis, rhinitis, and pruritus by inhibiting the expression‎ of IgE-switching cytokine IL-4 and proinflammatory cytokines IL-1β and TNF-α via the regulation of NF-κB and AP-1 activation.

 Key words Lactobacillus plantarum K-1, allergy, scratching behavior, passive cutaneous anaphylaxis

INTRODUCTION

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to reduced exposure to microbial stimuli (Guarner et al., 2006). It was supported by the finding that non-allergic children exhibit intensive colonization of aerobic bacteria or Bacteroides in their indigenous intestinal flora (Kallionmaki et al., 2001). Normal intestinal microflora consist of > 500 bacterial species and reach their highest concentrations in the terminal ileum, cecum and colon. Intestinal microflora produce toxic sources, such as gram-negative bacterial endotoxins, and harmful enzymes, such as β-glucuronidase and tryptophanase, which produce cytotoxic or carcinogenic agents. Cytotoxins and endotoxins are potent stimuli of innate immune responses, induce pro-inflammatory and IgE-inducing cytokines in colonic epithelial cells, and cause various diseases, such as colitis, carcinoma, anaphylaxis, and hypersensitivity. Therefore, researchers attempted to utilize symbiotic microorganisms in the treatment of immune disorders, such as anaphylaxis, pruritus, rhinitis, and atopic dermatis, and several in vitro, in vivo and clinical experiments have demonstrated the role of microorganisms as immune regulators (Majamaa and Isoauri, 1997; Isolauri, 2001; Ogden Bielory, 2005).

Lactic acid bacteria (LAB) are gram-positive, non-spore forming, non-respiring cocci or rods that ferment carbohydrates and produce lactic acid as the main product. (Simon and Gorbach, 1984; Collins and Gibson, 1999; Cho et al, 2006). The common LAB genera in fermented foods, such as cheese, yogurt, and the traditional Korean food, Kimchi, and intestinal microflora, are Lactobacillus sp., Lactococcus sp., Leuconostoc sp., Pediococcus sp., Enterococcus sp., Streptococcus sp., and Bifidobacterium sp. These LAB are safe microorganisms that improve any disturbances of indigenous microflora (Campieri and Gionchetti, 1999; Perdigon et al., 1991), ameliorate the development of beneficial microflora (Collins and Gibson, 1999), have anticolitic effects (Campieri and Gionchetti, 1999; Lee et al., 2010), and induce non-specific activation of the host immune system (Perdigon et al., 1991). Recently, the enhanced presence of LAB in the intestinal microbiota has been correlated with atopy preval‎ence (Majamaa and Isoauri, 1997; Kaillomaki et al., 2003). There is insufficient but very promising evidence supporting the addition of LAB to foods for the prevention and treatment of allergic diseases, especially atopic dermatitis. LAB also potentiate tolerance induction via the sublingual route as adjuvants for sublingual allergy vaccines by increasing IL-12 and IL-10 expression‎ in mice (van Overtvelt et al., 2010). Of these LAB, L. plantarum isolated from Kimchi, a traditional Korean

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food, reduced allergen-induced, airway hyper-responsiveness and enhanced the expression‎ of regulatory factors, such as Foxp3 and IL-10, in intestinal laminar propria cells (Hong et al., 2010). L. plantarum isolated from Kimchi was also found to inhibit the anti-scratching behavioral effect induced by histamine by inhibiting IL-4 expression‎ (Jang et al., 2011). However, its antiallergic effect remains to be thoroughly studied. Therefore, we isolated L. plantarum K-1, which most potently inhibited AP-1 and NF-κB activation in phorbol 12'-myristate 13'-acetate (PMA)-stimulated RBL-2H3 cells, from Kimchi, and its inhibitory activity against scratching behavior and passive cutaneous anaphylaxis (PCA) reaction in mice.

MATERIALS AND METHODS Materials Dulbecco’s modified Eagles medium (DMEM), fetal bovine serum, dinitrophenol-human serum albumin (DNP-HSA), histamine, phorbol 12'-myristate 13'-acetate (PMA) and Evans blue dye were purchased from Sigma Co. (St. Louis, MO, U.S.A.). Azelastine was donated by Dr. Nam-Jae Kim, an adjunct professor at Kyung Hee University. Enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D systems (Minneapolis, MN, USA). MRS was purchased from BD Co. (Sparks, MD, U.S.A.). Fifty LABs were isolated from kimchi according to the previous method of Lim and Im (2009). Animals Male ICR and BALB/c mice (20 - 22 g, 5 weeks old) were obtained from the Charles River Orient Experimental Animal Breeding Center (Seoul, Korea). All animals were housed in wire cages at 20-22°C, relative humidity of 50±10%, air ventilation frequency of 15-20 times/h and 12-h illumination (07:00-19:00; intensity, 150-300 Lux), fed standard laboratory chow (Charles River Orient Experimental Animal Breeding Center, Seoul, Korea), and allowed water ad libitum. All experiments were performed in accordance with the NIH and Kyung Hee University guides for Laboratory Animals Care and Use and approved by the Committee for the Care and Use of Laboratory Animals in the College of Pharmacy, Kyung Hee University.

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Measurement of Passive Cutaneous Anaphylasis (PCA) Reaction An IgE-dependent cutaneous reaction was measured according to the previous method of Choo et al. (2003). The male ICR mice were intradermally injected, with 10 μg of anti-DNP IgE, into each of two dorsal skin sites that had been shaved 48 h earlier. The sites were outlined with a water-insoluble red marker. Forty-eight hours later each mouse received an injection of 200 μl of 3% Evans blue in PBS, containing 200 μg of DNP-HAS, via the tail vein. LP (1 × 109 CFU and 1 × 1010 CFU/mouse) and azelastine (10 mg/kg) was administered once a day for 3 days. DNP-HSA injection was injected 1 h after the final administration of LP and azelastine. Thirty min after the DNP-HSA injection, the mice were sacrificed, their dorsal skins removed and the pigmented area measured. After extraction with 1 ml of 1.0 M KOH and 4 ml of a mixture of acetone and 0.2 M phosphoric acid (13:5), the amount of dye was determined colorimetrically at 620 nm.

Assay of Scratching Behavioral Frequency in Mice Male BALB/c mice were placed in acrylic cages (22 × 22 × 24 cm) for about 10 min to become acclimatized. The behavioral experiments were performed according to the method of Shin et al. (2007). The rostral part of the skin on the back of the mice was clipped, and 50 μg/50 μl of compound 48/80 (dissolved in saline) intradermally injected into each mouse. Control mice received a saline injection in the place of the compound 48/80. Immediately after the intradermal injection, the mice (one animal/cage) were put back into the same cage and the scratching behaviors recorded using an 8-mm video camera (SV-K80, Samsung, Seoul, Korea) under unmanned conditions. Scratching of the injected site with the hind paws was counted and compared with that of other sites, such as the ears. Each mouse was used for only one experiment. The mice generally showed several scratches per second, and a series of these behaviors was counted as one incident of scratching over a 60 min period. LP (1×109 CFU and 1x1010 CFU/mouse) and azelastine (10 mg/kg) was administered once a day for 3 days. The final administration of the test agents were performed 1 h before the scratching agent.

Measurement of Vascular Permeability

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The increase in vascular permeability caused by scratching agents was assessed as reported by Choo et al. (2003).. After the intradermal injection of 300 μg/50 μl of histamine into the rostral part of the back of each mouse, 0.2 ml of 1% saline solution of Evans blue dye was injected intravenously. LP (1 × 109 CFU/mouse and 1 × 1010 CFU/mouse) and azelastine (10 mg/kg) was administered once a day for 3 days. Mice were sacrificed by cervical dislocation 60 min after the final administration of test agents and the scratching agent-injection site was excised. The skin specimen was dissolved in 1 ml of 1 M KOH solution by overnight incubation, and 4 ml of 0.2 M phosphoric acid solution-acetone (5:13) mixture was added. After shaking, the precipitates were filtered off and the amount of dye was measured colorimetrically at 620 nm.

Enzyme-linked Immunosorbent Assay (ELISA) and Immunoblot For the assay of cytokines in skin tissues by ELISA, histamine-induced skin tissue specimens were homogenized in ice-cold lysis buffer (10 mM Tris, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.05% Nonidet P-40, 1 mM EGTA, 1:100 protease inhibitor cocktail, and 1:100 phosphatase inhibitor cocktail). Lysed specimens were centrifuged at 2,700 × g for 10 min at 4oC. The supernatant containing the cytosol was further centrifuged at 20,800 × g for 15 min at 4oC to obtain the cytosolic fraction. The nuclei in the pellet were washed 3 times by gentle resuspension in wash buffer (10 mM PIPES, pH 6.8, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 25 mM NaCl, 1:100 protease inhibitor cocktail and 1:100 phosphatase inhibitor cocktail) and centrifugation at 2,700 × g for 5 min at 4oC [19]. The supernatants (50 μl) were transferred to 96-well ELISA plates, and the concentrations of IL-4 and TNF-α were then determined using commercial ELISA kits (Pierce Biotechnology, Inc., Rockford, IL, U.S.A.). For the assay of cytokines in RBL-2H3 cells by ELISA, the cells (2 × 105 cells) previously cultured in DMEM were treated with 20 nM PMA. The cells (1.8 ml) were exposed to 0.2 ml of the test agents (0.2 × 109 or 1 × 109 CFU of heated LP dissolved in 0.5% dimethyl sulfoxide) for 4 h, followed by treatment with 0.2 ml DNP-HSA (1 μg/ml) for 40 min at 37oC. The supernatant (50 μl) was transferred to 96-well ELISA plates, and the IL-4 ad TNF-α concentrations then determined using commercial ELISA Kits.

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For the immunoblot analysis of transcription factors, phospho-p65, phospho-c-Jun (p-AP-1), p65 NF-κB, c-jun (AP-1) in the tissues and RBL-2H3 cells were performed as previously reported (Ryu et al., 2011). The protein fractions of each the lysate were subjected to electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel and then transferred to a nitrocellulose membrane. Phospho-p65, phospho-c-Jun, p65 NF-κB and c-jun were assayed with the corresponding antibodies. Immunodetection was carried out using an enhanced chemiluminescence detection kit (Thermo Fisher Scientific Inc., Rockford, IL, U.S.A.).

Statistical Analysis All data were expressed as the mean ± standard deviation, with statistical significance analyzed using one-way ANOVA followed by a Student-Newman-Keuls test.

RESULTS Inhibitory Effect of L. plantarum K-1 on the Activation of NF-κB and AP-1 in PMA-stimulated RBL-2H3 Cells To isolate anti-allergic LAB, we isolated 50 LABs from kimchi and investigated their inhibitory effect against the expression‎ of IL-4 and TNF-α and the activation of their transcription factors NF-κB and AP-1 in PMA-stimulated RBL 2H3 cells (Fig. 1). Treatment with PMA increased proinflammatory cytokine TNF-α and IgE-switching cytokine IL-4 expression‎. Furthermore, it activated transcription factor NF-κB and AP-1, which regulates the expression‎ of those cytokines. Of isolated LABs, L. plantarum K-1 most potently inhibited the expression‎ of those cytokines. At a concentration of 1 × 1010

L. plantarum K-1 was found to inhibit AP-1 and NF-kB activation in basophil cells. Therefore, the PCA reaction-inhibitory effect of L. plantarum K-1 in mice was measured (Fig. 2). The PCA CFU/ml. L. plantarum K-1 inhibited IL-4 and TNF-α expression‎ by 92% and 28%, respectively. LP also potently inhibited NF-κB and AP-1 activation.

Inhibitory Effect of L. plantarum K-1 on IgE-antigen Complex–induced Passive Cutaneous Anaphylaxis in Mice

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reaction was induced by an injection of IgE and antigen, with L. plantarum K-1 administered orally once a day for three days prior to the challenge with antigen. The IgE-antigen complex potently induced the PCA reaction. LP showed potent inhibition against the PCA reaction. It was inhibited by 85.7% at a dose of 1x1010 CFU/mouse.

Anti-scratching Behavioral Effect of L. plantarum in Histamine-treated Mice The inhibitory effect of L. plantarum K-1 against histamine-induced scratching behavior in mice was investigated (Fig. 3A). L. plantarum K-1 potently inhibited the scratching behavior induced by histamine. ). L. plantarum K-1 inhibited it by 58.9% at a dose of 1×1010 CFU/mouse compared to the effects in the control group. The agent also inhibited the scratching behavior induced by histamine. When histamine was used as an inducer for scratching, the histamine increased vascular permeability as well as induced scratching behavior. L. plantarum K-1 significantly inhibited vascular permeability induced by histamine (Figure 3B). The inhibitory activity of L. plantarum K-1 against vascular permeability was in proportion to its inhibition against scratching behavior. To confirm‎ the anti-scratching behavioral effect of L. plantarum K-1, we investigated its inhibitory effect against IL-4 and TNF-α protein expression‎ and NF-κB and AP-1 activation as well as IgE level in mouse skin stimulated with histamine (Fig. 4). Histamine increased the IgE-switching cytokine IL-4 and proinflammatory cytokine IL-1β and TNF-α protein expression‎ by 1.9-fold, 1.7-fold, and 2.1-fold, respectively (Fig. 4). Histamine also increased the IgE level 1.9-fold. L. plantarum K-1 potently inhibited histamine-induced cytokine production: at 1×1010

Mast cells and basophils are well-known critical participants in various biological processes of allergic diseases (Maintz and Novak, 2007; Reich and Szepietowski, 2007). These cells express CFU/mouse IL-4, IL-1β and TNF-α expression‎ was inhibited by 88.9%, 88.6%, and 98.9%, respectively. L. plantarum K-1 also inhibited IgE level increased by histamine by 85.3%. It also inhibited histamine-induced activation of the transcription factors, NF-κB and c-jun, which regulate TNF-α and IL-4 expression‎, respectively (Fig. 5).

 DISCUSSION

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surface membrane receptors, with high affinity and specificity for IgE. The interaction of antigen-bound IgE in surface membrane receptors induces the release of histamine, prostaglandins, leukotrienes and cytokines (Kuraishi et al., 1995), and finally cause pruritus, inflammation, rhinitis, atopic dermatitis, asthma and food allergies. These allergic diseases are now rapidly increasing chronic health problem in most countries (Mitre and Nutman, 2006). Antiallergic agents, such as anti-histamines, steroids, immunosuppressants, and quercetin, have been used against allergic diseases (Simons, 1992; Schafer-Korting et al., 1996; Sakuma et al., 2001), but improving these diseases is very difficult. Therefore, LAB have been advanced for allergic diseases, and their effectiveness has been given increasing attention.

In the present study, L. plantarum K-1 potently inhibited IgE-induced PCA reaction in mice. LP also inhibited the expression‎ of proinflammatory cytokines TNF-α and IL-1β and the IgE-switching cytokine IL-4 as well as the activation of their transcription factor NF-κB and AP-1 in HMC-1 cells. Furthermore, L. plantarum K-1 inhibited histamine-induced scratching behavior as LAB are recognized as beneficial microorganisms and have been demonstrated to exert preventive and therapeutic effects on Th2-bias diseases such as atopic dermatitis or food allergies (Kaillomaki et al., 2003; van Overtvelt et al., 2010). Fermented foods, such as yogurt, cheese, and Kimchi, are considered to be good sources of microorganisms; a variety of LAB strains have been detected in those foods (Cho et al., 2006) and are known to regulate host defense mechanisms (Lee et al., 2005; Cho et al., 2007). Particularly, L. plantarum isolated from Kimchi may have beneficial effects in allergic diseases, such as asthma, by enhancing the expression‎ of regulatory factors, such as Foxp3 and IL-10, in intestinal laminar propria cells (Cho et al., 2006; Hong et al., 2010). L. plantarum inhibited scratching behaviors caused by histamine or compound 48/80 via inhibiting IL-4 expression‎ in mice (Jang et al., 2011). L. rhamnosus GG was noted to exert suppressive effects on asthma, and this may be correlated with increased numbers of Treg cells (Feleszko et al., 2007). L. paracasei was used to treat human patients suffering from allergic rhinitis (Pent and Hsu, 2005). Those results demonstrated that Lactobacillus species might modulate dendritic cells or T cell-mediated responses in vitro by boosting the production of immunosuppressive cytokines, IL-10 and TGF-β (von der Eeid et al., 2001; Khoury et al., 1992).

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well as vascular permeability in mice. L. plantarum K-1 inhibited the expression‎ of IL-4, TNF-α, IL-1β, and IgE in mouse skin stimulated with histamine. Based on those findings, L. plantarum K-1 may improve scratching by regulating the expression‎ of IL-4, which induces IgE production and inflammatory diseases, such as colitis, by suppressing the expression‎ of proinflammatory cytokines, IL-1β and TNF-α via NF-κB and AP-1 activation. Furthermore, L. plantarum K-1 may improve allergic diseases, such as anaphylaxis, atopic dermatitis, rhinitis, and pruritus.

REFERENCES Campieri, M., and Gionchetti, P. Probiotics in inflammatory bowel disease: New insight to pathogenesis or a possible therapeutic alternative. Gastroenterology 116, 1246-1260 (1999). Cho, J., Lee, D., Yang, C., Jeon, J., Kim, J., and Han, H. Microbial population dynamics of kimchi, a fermented cabbage product. FEMS Microbiol. Lett. 257, 262-267 (2006). Cho, Y.R., Chang, J.Y., and Chang, H.C. Production of gammaaminobutyric acid (GABA) by Lactobacillus buchneri isolated from kimchi and its neuroprotective effect on neuronal cells. J. Microbiol. Biotechnol. 17, 104-109 (2007). Choo, M.K., Park, E.K., Han, M.J., and Kim, D.H. Antiallergic activity of ginseng and its ginsenosides. Planta Med. 69, 518-522 (2003). Collins, M.P., and Gibson, G.R. Probiotics, prebiotics, and synbiotics: approaches for modulating the microbial ecology of the gut. Am. J. Clin. Nutr. 69, s1052-s1057 (1999). Feleszko, W., Jaworska, J., Rha, R.D., Steinhausen, S., Avagyan, A., Jaudszus, A., Ahrens, B., Groneberg, D.A., Wahn, U., and Hamelmann, E. Probiotic-induced suppression of allergic sensitization and airway inflammation is associated with an increase of T regulatory-dependent mechanisms in a murine model of asthma. Clin. Exp/ Allergy. 37, 498-505 (2007). Guarner, F., Bourdet-Sicard. R., Brandtzaeg, P., Gill, H.S., McGuirk, P., van Eden, W, Versalovic, J., Weinstock, J.V., and Rook, G.A. Mechanisms of disease: the hygiene hypothesis revisited. Nat. Clin. Pract. Gastroenterol. Hepatol. 3, 275-284 (2006).

Hong, H.J., Kim, E., Cho, D., and Kim, T.S. Differential suppression of heat-killed Lactobacilli

isolated from kimchi, a Korean traditional food, on airway hyper-responsiveness in mice. J. Clin. Immunol. 30, 449-458 (2010). Isolauri, E. Probiotics in the prevention and treatment of allergic disease. Pediatr. Allergy Immunol. 2001, 12 Suppl 14, 56-59. Jang, S..E, Hyun, Y..J, Trinh, H.T., Han, M.J., and Kim, D.H. Anti-scratching behavioral effect of Lactobacillus 16. plantarum PM008 isolated from kimchi in mice. Immunopharmacol Immunotoxicol. (2011). [Epub ahead of print]. Kaillomaki, M., Salminen, S., Poussa, T., Arvilommi ,H., and Isolauri, E. Probiotics and prevention of atopic disease: 4-year follow-up of a randomized placebo-controlled trial. Lancet 361, 1869-1871 (2003). Kalliomäki, M., Kirjavainen, P., Eerola, E., Kero, P., Salminen, S., and Isolauri, E.. Distinct patterns of neonatal gut microflora in infants in whom atopy was and was not developing. J. Allergy Clin. Immunol. 107, 129-134 (2001). Khoury, S.J., Hancock, W.W., and Weiner. H.L. Oral tolerance to myelin basic protein and natural recovery from experimental autoimmune encephalomyelitis are associated with downregulation of inflammatory cytokines and differential upregulation of transforming growth factor beta, interleukin 4, and prostaglandin E expression‎ in the brain. J. Exp. Med. 176, 1355-1364 (1992). Kuraishi, Y., Nagasawa, T., Hayashi, K., and Satoh, M. Scratching behavior induced by pruritogenic but not algesiogenic agents in mice. Eur. J. Pharmacol. 275, 229-233 (1995). Lee, J., Hwang, K.T., Heo, M.S., Lee, J.H., and Park, K.Y. Resistance of Lactobacillus plantarum KCTC 3099 from Kimchi to oxidative stress. J. Med. Food. 8, 299-304 (2005). Lee, I.A., Bae, E.A., Lee, J.H., Lee, H., Ahn, Y.T., Huh, C.S., and Kim, D.H. Bifidobacterium longum HY8004 attenuates TNBS-induced colitis by inhibiting lipid peroxidation in mice. Inflamm. Res. 59, 359-368 (2010). Lim, S.M., and Im, D.S. Screening and characterization of probiotic lactic acid bacteria isolated from Korean fermented foods. J. Microbiol. Biotechnol. 19, 178-186 (2009). Maintz, L., and Novak, N. Histamine and histamine intolerance. Am. J. Clin. Nutr. 85, 1185-1196 (

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Mitre, E., and Nutman, T.B. Basophils, basophilia and helminth infections. Chem. Immunol. Allergy 90, 141-156 (2006). Ogden, N.S., Bielory, L. Probiotics: a novel approach in the treatment and prevention of pediatric atopic disease. Curr. Opin. Allergy Clin. Immunol. 2005, 5, 179-184. Peng, G.C., and Hsu, C.H. The efficacy and safety of heat-killed Lactobacillus paracasei for treatment of perennial allergic rhinitis induced by house-dust mite. Pediatr. Allergy Immunol. 16, 433-438 (2005). Perdigon, G., de Jorrat, W.E.B., de Petrino, S.F., Valerde de Budeguer, M. Effect of oral administration of Lactobacillus casei on various biological functions of the host. Food Agric. Immunol. 3, 93-102 (1991). Reich, A., and Szepietowski, J.C. Mediators of pruritus in psoriasis. Mediators Inflamm. 2007, 64727 (2007). Ryu, K.R., Choi, J.Y., Chung, S., and Kim, D.H. Anti-scratching behavioral effect of the essential oil and phytol isolated from Artemisia princeps Pamp. in mice. Planta Med. 77, 22-26 (2011). Sakuma, S., Higashi, Y., Sato, N., Sasakawa, T., Sengoku, T., Ohkubo, Y., and Amaya, T. Tacrolimus suppressed the production of cytokines involved in atopic dermatitis by direct stimulation of human PBMC system. (Comparison with steroids). Int. Immunopharmacol. 1, 1219-1226 (2001). Schafer-Korting, M., Schmid, M.H., and Korting, H.C. Topical glucocorticoids with improved risk-benefit ratio. Rationale of a new concept. Drug Safety 14, 375-385 (1996). Shin, Y.W., Bae, E.A., Lee, B., Lee, S.H., Kim, J.A., Kim, Y.S., Kim, D.H. In vitro and in vivo antiallergic effects of Glycyrrhiza glabra and its components. Planta Med

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LEGENDS

Fig. 1. Inhibitory effect of Lactobacillus plantarum K-1 on the expression‎ of IL-4 (A), TNF-α (B) and their transcription factors, AP-1 and NF-κB, (C) in PMA-stimulated RBL-2H3 cells. RBL-2H3 cells (2 × 105) was stimulated with 20 nM PMA (PM) and then heat-inactivated L. plantarum K-1 (LPL, 0.2 × 109CFU/well: LPH, 1 × 109 CFU/well) was treated. Normal group (NOR) was treated with vehicle alone instead of L. plantarum K-1. TNF-α and IL-4 were assayed by ELISA and NF-κB and AP-1 were determined by immunoblot analysis. Mean ± SD (n=5). # p < 0.05 vs. normal control group. * p < 0.05 vs. PMA-treated group. Fig. 2. Inhibitory effect of Lactobacillus plantarum K-1 on IgE-induced passive cutaneous anaphylaxis reaction in mice. The passive cutaneous anaphylaxis reaction in two dorsal skin sites of mice was induced by an intradermal injection of anti-DNP-HSA. K-1 and azelastine were orally administered once a day for three days. The amounts of extravasated Evan blue from the dorsal skin (1 × 1 cm) were measured 1 h after the final administration of test agents. NOR, normal group treated with vehicle alone; CON, control treated with IgE-antigen complex alone; LPL, 1 × 109 CFU of L. plantarum K-1 with IgE-antigen complex; LPH, 1 × 1010 CFU of L. plantarum K-1 with IgE-antigen complex; AZ, 10 mg/kg azelastine with IgE-antigen complex. Mean ± SD (n=5). # p < 0.05 vs. normal control group. *

Fig. 3. Effect of Lactobacillus plantarum K-1 on histamine-induced scratching behavior in mice. (A) Effect on scratching behavior. The scratching behaviors in the normal control group treated with saline alone and in the histamine-treated groups with and without test agents were counted for 1 h. L. plantarum K-1 (1 × 10 p < 0.05 vs. IgE-antigen complex-treated group.

9 CFU/mouse and 1 × 1010 CFU/mouse) and azelastine (10 mg/kg) was administered once a day for three days. The final administration of test agents were performed 1 h before histamine administration. NOR, normal group; CON, control treated with IgE-antigen complex alone; LPL, 1×109 CFU of L. plantarum K-1 with IgE-antigen complex; LPH, 1×1010 CFU of L. plantarum K-1 with IgE-antigen complex; AZ10, 10 mg/kg azelastine with histamine. (B) Effect on vascular permeability. The vascular permeability was increased by histamine in mice. Mice were treated with or without the oral administration of test agents 1 h before the intradermal injection of 300 μg/50 μL of histamine into the skin on the backs of mice. In the vascular permeability assay, the amount of Evan blue extravasated from the dorsal skin (1×1 cm) of mice was measured. Mean ± SD (n=5). # p < 0.05 vs. normal control group.*p < 0.05 versus histamine-treated group.

Fig. 4. Inhibitory effects of Lactobacillus plantarum K-1 on the protein expressions of TNF-α (A) and IL-4 (B), IL-1β (C) and IgE (D) in histamine-induced mouse skin tissues. TNF-α (a) and IL-4 (b), IL-1β (c) and IgE (d) were assayed by ELISA. L. plantarum K-1 and azelastine were orally administered to mice once a day for three days: NOR, normal group; CON, control treated with IgE-antigen complex alone; LPL, 1x109 CFU of L. plantarum K-1with IgE-antigen complex; LPH, 1x1010 CFU of L. plantarum K-1 with IgE-antigen complex; AZ, 10 mg/kg azelastine with histamine. Mean ± SD (n=5). # p < 0.05 vs. normal control group. * p < 0.05 vs. histamine-treated group.

Fig. 5. Inhibitory effects of Lactobacillus plantarum K-1 on the activation of transcription factors NF-κB and c-jun in histamine-induced mouse skin tissues. NF-κB, c-jun and β-actin were determined by immunoblot analysis. L. plantarum K-1and azelastine were orally administered to mice once a day for three days: NOR, normal group; CON, control treated with IgE-antigen complex alone; LPL, 1x109 CFU of L. plantarum K-1 with IgE-antigen complex; LPH, 1 × 1010 CFU of L. plantarum K-1 with IgE-antigen complex; AZ, 10 mg/kg azelastine with histamine.

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