KR101094194B1 - Lactic acid bacteria for treating and/or preventing atopic dermatitis and composition containing these bacteria - Google Patents

Abstract

The present invention relates to lactic acid bacteria useful for the treatment and / or prevention of atopic dermatitis and compositions containing them. The lactic acid bacteria for treating and / or preventing atopic dermatitis of the present invention are Leuconostoc citreum KACC91035, Leukonostoc mesenteroides subs. brevis) lactic acid bacteria selected from the group consisting of KCTC 3498, the composition of the present invention containing such lactic acid bacteria or cultures thereof has an excellent effect in the treatment and / or prevention of atopic dermatitis.

Atopic dermatitis, lactic acid bacteria, IgE, IFN-γ

Description

Lactic acid bacteria for treating and / or preventing atopic dermatitis and composition containing these bacteria}

1 is a graph showing the result of the ovalbumin-specific IgE concentration of mice sensitized with ovalbumin was changed by the lactic acid bacteria of the present invention. O.D. stands for optical density.

Figure 2 is a graph showing the result of the IFN-γ concentration of the mouse sensitized with ovalbumin changed by the lactic acid bacteria of the present invention.

Figure 3 is a graph showing the result of the ovalbumin-specific IgE concentration of the mice sensitized with ovalbumin after the first lactic acid bacteria were changed by the lactic acid bacteria of the present invention. O.D. means optical density.

4 is a graph showing the result of the concentration of IFN-γ in mice sensitized with ovalbumin after the lactic acid bacteria were first administered by the lactic acid bacteria of the present invention.

In Figures 1 to 4, the control (control) is the result of administration of the ovalbumin only antigen, 1 is the result of the administration of leukonostock citrium KACC91035, 2 is the administration of leukonostock mesenteroides subspecies mesenteroides KCTC 3100 3 is the result of administration of Lactobacillus brevis KCTC 3498. * Means P <0.05 for t-test, ** means P <0.01 for t-test, and *** means P <0.005 for t-test.

The present invention relates to lactic acid bacteria for treating and / or preventing atopic dermatitis and to compositions containing such lactic acid bacteria or cultures thereof.

Atopic dermatitis is a disease in which the skin is chronically inflamed by the hypersensitivity of the skin to triggers that are not harmful to normal people. In other words, atopic dermatitis is known to be caused by an overreaction of the immune system to certain substances such as bacteria, viruses, fungi, pollen, foods, artificial chemicals, and the like.

T cells are involved in this immune response, and the cytokines secreted by T cells are generally divided into two types, Th1 cytokines and Th2 cytokines. Th1 cytokines are involved in cell-mediated immune responses, and Th2 cytokines are responsible for antibody immune responses. Both atopic dermatitis and other allergic disorders can disrupt the balance of these two cytokines, leading to interleukin-4 (IL-4), Overproduction of Th2 cytokine secretion, such as IL-5, IL-13, and the like, decreases the secretion of Th1 cytokines, such as interferon-γ (IFN-γ), which are antagonically secreted by Th1 cells (Jujo K. et al. , Decreased interferone gamma and increased interleukin-4 production in atopic dermatitis promotes IgE synthesis.J Allergy Clin Immunol 1992; 90: 323-31 and Tang ML., Et al ., Interleukin-4 and interferone-γ production in atopic and non-atopic children with asthma.Clin Exp Allergy 1995; 25: 515-21).

In addition, Staphylococcus aureus clusters in the skin of most patients with atopic dermatitis, which is about 10 ⁷ CFU / ㎠, 200 times higher than normal people. Because of these bacteria, immunoglobulin E (IgE) continues to be produced, and the ceramidase secreted by these bacteria breaks down ceramide, which keeps the subcutaneous moisturizing ability, leading to itching. Worsen. Once the atopic dermatitis start due to facilitate a Th2 cytokine which has these bacteria IL-4 from sticking to the skin staphylococci is proliferate beyond catch surface (Donald YM., Etc., Infection in atopic dermatitis. Curr Opin Pediatr 2003; 15: 399-404 and Chos SH. Et al., Preferential binding of Staphylococcus aureus to skin sites of Th2-mediated inflammation in a murine model.J Invest Dermatol 2001; 116: 658-63).

Steroids, antihistamines, antibiotics, etc. are used to treat atopic dermatitis, but steroids such as corticosteroids have anti-inflammatory and immunosuppressive effects, which are excellent but prolonged severe skin side effects. Concerns are high and bacterial infections can occur. In addition, antihistamines may temporarily relieve itching, but may cause various side effects when taken for a long time, and antibiotics are used to treat secondary bacterial infections, such as staphylococcus, but are not a fundamental treatment.

On the other hand, there was a strong correlation between the composition of intestinal bacteria and the prevalence of allergic diseases (Bjorksten B et al., The intestinal micoroflora in allergic Estonian and Swedish 2-year-old children, Clin Exp Allergy 1999; 29 (3)). (Refer to 342-346). Research is being conducted to prevent allergic diseases using bacteria that are harmless to humans. For example, studies have reported that some lactic acid bacteria are effective in preventing and / or treating atopic diseases that are immune imbalances of Th1 cytokines and Th2 cytokines by promoting the secretion of Th1 cytokines (Aattouri N. et al., Oral ingestion of lactic acid bacteria by rats increases lymphocyte proliferation and interferon-gamma production, British Journal of Nutrition 2002; 87: 367-373, Repa A. et al., Mucosal co-application of lactic acid bacteria and allergen induces counter-regulatory immune responses in a murine model of birch pollen allergy, Vaccine 2003; 22: 87-95 and Gill HS et al., Enhancement of nutural and acquired immunity by Lactobacillus rhamnosus, Lactobacillus acidophilus and Bifidobacterium lactis, British Journal of Nutrition 2000; 83: 167-176).

Accordingly, the present invention provides a lactic acid bacterium that can treat and / or prevent atopic dermatitis by promoting the secretion of Th1 cytokines and suppressing the secretion of Th2 cytokines to resolve the imbalance between Th1 and Th2 cytokine secretions. It is to provide a composition containing such lactic acid bacteria or cultures thereof.

In order to achieve the above technical problem, the present invention is a Leuconostoc citreum KACC91035, Leukonostoc mesenteroides subsp. Mesenteroides KCTC 3100 and Lactobacillus brevis (Lactobacillus brevis) Provided are lactic acid bacteria for the treatment and / or prophylaxis of atopic dermatitis selected from the group consisting of KCTC 3498.

The present invention is also directed to the treatment of atopic dermatitis containing any one or more lactic acid bacteria or cultures thereof selected from the group consisting of leukonostock citrium KACC91035, leukonostock mesenteroides subspecies mesenteroides KCTC 3100 and Lactobacillus brevis KCTC 3498 or Provided is a prophylactic composition.

The lactic acid bacteria of the present invention are useful for the treatment and prevention of atopic dermatitis by increasing the secretion of the Th1 cytokine IFN-γ as well as reducing the amount of IgE.

Hereinafter, the lactic acid bacteria for treating and / or preventing atopic dermatitis of the present invention and a composition containing such lactic acid bacteria or a culture thereof will be described in more detail.

The present invention is a Leuconostoc citreum (Accession No. KACC91035), Leukonostoc mesenteroides subspecies Mesenteroides (Accession no. 3498) is provided for the treatment and / or prophylaxis of atopic dermatitis selected from the group consisting of.

In the present invention, the leukonostock citrium was used by the present inventors the strain deposited on March 7, 2003, the accession number KACC91035 to the Korea Agricultural Microorganisms Conservation Center (KACC), the leukonostock mesenteroides subspecies mesenteroi Death KCTC 3100 and Lactobacillus brevis KCTC 3498 were purchased from the Institute of Biotechnology Gene Bank (KCTC).

The therapeutic or prophylactic effect of the lactic acid bacteria of the present invention for atopic dermatitis can be confirmed by various methods. For example, in order to evaluate the therapeutic effect on atopic dermatitis, mice are sensitized with an allergen ovalbumin to make atopic dermatitis, and then the lactic acid bacteria is administered to evaluate the improvement of atopic dermatitis, To evaluate the prophylactic effect against atopic dermatitis, the amount of IgE and IFN-γ produced by sensitization with ovalbumin after administration of the lactic acid bacterium was compared and compared to the control group to treat or prevent the atopic dermatitis. You can check.

The lactic acid bacteria of the present invention, Leukonostock Citrium KACC91035, Leukonostock Mesenteroides subspecies Mesenteroides KCTC 3100 and Lactobacillus brevis KCTC 3498 showed excellent effects in the treatment and prophylactic evaluation trials for such atopic dermatitis.

The present invention also relates to atopic dermatitis containing any one or more lactic acid bacteria or cultures thereof selected from the group consisting of the aforementioned leukonostock citrium KACC91035, leukonostock mesenteroide subspecies mesenteroides KCTC 3100 and Lactobacillus brevis KCTC 3498. Provided are compositions for treatment or prophylaxis.

The composition of the present invention may be formulated in the form of powder, granules, tablets, capsules or beverages by mixing with appropriate carriers, excipients, auxiliary active ingredients and the like in addition to the lactic acid bacteria. That is, the composition of the present invention may be formulated into powders and granules using known methods such as freeze drying using a standard carrier, and also in tablet or capsule form using conventional tablet and encapsulation methods. Can be. In addition, the composition of the present invention may be enterically coated and commercialized using known methods to reach the small intestine after passing through the stomach and rapidly release the active ingredient microorganism into the intestine. Suitable carriers, excipients and diluents for use in the formulation of the compositions of the invention include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, tragacanth rubber, gelatin, calcium silicate, Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl and propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. Lubricants, wetting agents, emulsifiers and suspending agents, preservatives, sweeteners or flavoring agents may be further included for the commercialization of the compositions of the present invention.

In addition, the composition of the present invention can be used in food and beverage, cosmetics, ointment, oral dosage form and the like for the treatment and / or prevention of atopic dermatitis.

Hereinafter, examples will be described in order to describe the present invention in more detail. However, embodiments according to the present invention may be modified in many different forms and should not be construed that the scope of the invention is limited to the embodiments described below. Embodiments of the present invention are provided to more fully explain the present invention to those skilled in the art.

Experimental Animal Preparation

Experimental animals were used to divide the 8 weeks old BALB / c male mice in the Orient Co., Ltd. divided into the control group and the experimental group. Solid feed and water were supplied unrestricted, giving 12 hours of day and night rhythms and adapted for 1 week at constant temperature and humidity.

Induction of Systemic Anaphylaxis in Mice

0.1% of ovalbumin (Ovalbumin, OVA, Sigma Co., Ltd.) was used as an antigen as a substance for inducing hypersensitivity reactions such as atopic dermatitis. 0.1% of ovalbumin was inoculated three times a week in the mouse abdominal cavity and sensitized. For one week after the last intraperitoneal administration, ovalbumin solution was instilled into the nasal cavity of the control and experimental mice.

<Administration of Lactic Acid Bacteria>

The lactic acid bacteria were each diluted in phosphate buffered saline (PBS) at a concentration of 2 × 10 ⁸ cells / ml and administered at 100 μl each for 2 days. For the evaluation of the prophylactic effect, a sample containing lactic acid bacteria was orally administered 1 week before the sensitization with ovalbumin, and for the evaluation of the therapeutic effect, 2 weeks after the sensitization with the ovalbumin until the separation of the serum or the extraction of the spleen Oral administration as well.

Separation of Serum for OVA-Specific IgE Measurements

The mice were anesthetized and blood was drawn from the heart. After the blood was collected, the blood was coagulated, and then serum was separated by centrifugation and stored at -20 ° C until analysis of IgE.

<Preparation of Splenocyte Suspensions for IFN-γ Measurement and Culture of Splenocytes>

The spleens of the mice were extracted and washed with RPMI-1640 (Invitrogen, USA) containing 10% fetal bovine serum (FBS). The spleen was crushed with a micro slide glass and filtered with a 0.40 μm nylon cell strainer. After centrifugation at 1000 rpm for 10 minutes, erythrocytes were destroyed with erythrocyte lysis buffer. Splenocytes were resuspended in 10% FBS RPMI-1640 after two centrifugations.

The resuspended splenocytes were seeded at 1 × 10 ⁶ cells / ml in a 24 well plate and then incubated with ovalbumin (1 mg / ml) in a 37 ° C., 5% CO ₂ incubator for 72 hours. After incubation, the supernatant was centrifuged and stored at -20 ° C until analysis.

<Measurement of IFN-γ and OVA-specific IgE by ELISA Method>

Measurement of interferon -γ (IFN-γ) was performed using the OPT EIA set (Pharmingen Inc., country of origin). Each well of a 96 well plate was coated with capture antibody at 4 ° C. overnight. Capture IgE antibody (2 μg / ml) was coated in the wells for the determination of OVA-specific IgE. 200 μl / well of PBS containing 10% FBS was added and blocked at room temperature for 1 hour. After washing three times to completely remove the blocking buffer, standard IgE (standard IgE), standard IFN-γ, and the sample were diluted appropriately, 100 μl were dispensed and left at room temperature for 2 hours. After washing 5 times, biotinylated detection antibody against IgE and IFN- [gamma] (Pharmingen, USA) and avidin were dispensed at 100 [mu] l and placed at room temperature for 1 hour. Biotin-attached ovalbumin was prepared after mixing diabolic with water-soluble biotin N hydroxysuccinimide ester-Water Soluble (Vector Laboratories, USA) and ovalbumin. After 7 washes, 100 μl of TMB substrate reagent was added, and after 30 minutes, 50 μl of 1M H ₂ SO ₄ was added. Optical density (OD) was measured at 450-570 nm with a microplate reader (Molecular Devices, USA).

<Statistical processing>

Significance test was determined by independent T test using SPSS 11.0.

<OVA-specific IgE measurement result>

The results of the evaluation of the therapeutic effect among the OD values of the OVA-specific IgE measured by the above experiments are shown in FIG. 1 and the results of the prophylactic effect are shown in FIG. 3. The lower the OD value measured in this experiment means that lactic acid bacteria inhibit the production of IgE. From these results, it can be seen that the lactic acid bacteria of the present invention are effective in the treatment and / or prevention of atopic dermatitis. As shown in Figures 1 and 3, lactic acid bacteria of the present invention reduced the amount of IgE compared to the control.

<IFN-γ measurement result>

Results of the evaluation of the therapeutic effect in the secretion amount of IFN- [gamma] measured by the above experiments are shown in FIG. 2, and the results of the prophylactic effect are shown in FIG. As shown in Figures 2 and 4, it can be seen that the lactic acid bacteria of the present invention increases the secretion amount of IFN-γ compared to the control group and from these results the lactic acid bacteria of the present invention can be used for the treatment and / or prevention of atopic dermatitis It can be seen that.

The composition of the present invention containing a lactic acid bacterium selected from the group consisting of leukonostock citrium KACC91035, leukonostock mesenteroides subspecies meccetoroides KCTC 3100 and Lactobacillus brevis KCTC 3498 and such lactic acid bacteria or cultures thereof It has the effect of treating and / or preventing atopic dermatitis by increasing the secretion of Th1 cytokines, which play a role in reducing the levels of IgE and inhibiting atopic dermatitis in patients with sexual dermatitis.

Claims (2)

Atopic composition containing lactic acid bacteria or cultures thereof selected from the group consisting of Leuconostoc citreum KACC 91035 and Leuconostoc mesenteroides subsp.mesenteroides KCTC 3100 Pharmaceutical composition for treating or preventing dermatitis. The method of claim 1, wherein the lactic acid bacterium or its culture is for treating or preventing atopic dermatitis that inhibits the production of immunoglobulin E (IgE) or promotes the secretion of interferon-gamma (IFN-γ). Pharmaceutical compositions.

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