Composition for preventing or treating wounds and promoting skin regeneration comprising lactobacillus reuteri nchbl-005 strain

Abstract

The present invention relates to a lactic acid bacteria formulation capable of healing wounds, comprising a Lactobacillus reuteri NCHBL-005 strain as an active ingredient. Specifically, a Lactobacillus reuteri NCHBL-005 strain, which is one of lactic acid bacteria formulations having a positive effect on the body, is cultured, and the Lactobacillus reuteri NCHBL-005 strain which can be safely used in the human body without toxicity and side effects can be used as a therapeutic formulation. More specifically, as a result of inducing a wound in a mouse fibroblast cell line by using a pipette tip and treating same with a Lactobacillus reuteri NCHBL-005 strain, it was confirmed that the migration and proliferation ability of the cell line was promoted, resulting in rapid wound recovery. The Lactobacillus reuteri NCHBL-005 strain was confirmed to promote the phosphorylation of p38 MAPK, ERK, and JNK, which are protein kinases involved in cell migration and proliferation ability and to promote wound healing in a manner dependent thereon. It was also confirmed that, when a wound was induced in the skin of a mouse and a wound site was directly treated with the Lactobacillus reuteri NCHBL-005 strain, wound healing was promoted compared to an untreated group.

Description

Composition for preventing or treating wounds and promoting skin regeneration, comprising Lactobacillus ruteri NCHBL-005 strain

[0001] The present invention was made under the support of the Ministry of Science and ICT under the research project number 1711184598, and the research management organization of the project is the National Research Foundation of Korea, the research project name is "Individual Basic Research", the research project title is "Study on the Disease Control Mechanism of Probiotics through Regulation of Node-Like Receptor Signaling", the project performing organization is Chonnam National University, and the research period is from 2022.03.01 to 2027.02.28.

[0002] The present invention relates to a composition for preventing or treating wounds and promoting skin regeneration, comprising a Lactobacillus reuteri NCHBL-005 strain, and more particularly, to a composition capable of exhibiting an effect for preventing or treating wounds and promoting skin regeneration by using a Lactobacillus reuteri NCHBL-005 strain that has the effect of enhancing the migration and proliferation of fibroblasts.

[0003] The skin, the largest tissue in the human body, is home to a diverse community of microorganisms, known as the skin microbiome. This microbial community plays a crucial role in the health and disease prevention of the host's skin, and is known to influence factors such as skin pH, moisture content, and the immune system. In particular, the skin microbiome's influence on the skin's immune response is known to induce an appropriate inflammatory response that protects the host skin from external bacteria, viruses, and other pathogens.

[0004] Meanwhile, lactic acid bacteria are known to play a crucial role in the skin microbiome. They are known to help maintain skin health by suppressing inflammation and irritation and regulating the skin immune system. Furthermore, lactic acid bacteria are known to strengthen the skin barrier, protecting the skin from external stressors and preventing moisture loss. Through these mechanisms, lactic acid bacteria are being applied in various skin conditions.

[0005] Wound healing is a process of tissue regeneration, including an inflammatory response, at the site of skin injury. This process can be divided into several stages, including hemostasis, inflammation, proliferation, and remodeling. Various internal and external factors are known to influence this process and contribute to wound healing. Recently, topical probiotics have been shown to be effective in wound healing by regulating the appropriate inflammatory response, preventing wound infection and accelerating healing.

[0006] However, the results on the protective effect on wound healing vary depending on the type of lactic acid bacteria, and the wound healing effect of Lactobacillus ruteri strains has not been reported to date, so additional research is needed in this regard.

[0007] Against this backdrop, the present inventors conducted research on the wound healing effect of the Lactobacillus ruteri strain, and as a result, they induced a wound in a mouse fibroblast cell line using a pipette tip and treated it with the Lactobacillus ruteri NCHBL-005 strain, and confirmed that the cell line's migration and proliferation ability was promoted, leading to rapid wound recovery, and that wound healing was promoted in a protein kinase MAPK-dependent manner.

[0008] Additionally, we confirmed that wounds were induced on the skin of mice and the Lactobacillus rue NCHBL-005 strain was directly applied to the wound site to aid in wound healing.

[0009] Accordingly, the purpose of the present invention is to provide a Lactobacillus reuteri NCHBL-005 strain deposited under accession number KCTC 15449BP, which has the effect of improving the migration and proliferation of fibroblasts.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating wounds, comprising as an active ingredient Lactobacillus reuteri NCHBL-005 strain, which has the effect of improving the migration and proliferation of fibroblasts.

[0011] Another object of the present invention is to provide a cosmetic composition for promoting skin regeneration, which comprises as an active ingredient Lactobacillus reuteri NCHBL-005 strain, which has the effect of improving the migration and proliferation of fibroblasts.

[0012] Another object of the present invention is to provide a health functional food composition for promoting skin regeneration, which contains as an active ingredient Lactobacillus reuteri NCHBL-005 strain, which has the effect of improving the migration and proliferation of fibroblasts.

[0013] Another object of the present invention is to provide a use of Lactobacillus reuteri NCHBL-005 strain for preventing or treating wounds and promoting skin regeneration.

[0014] The present invention relates to a composition for preventing or treating wounds and promoting skin regeneration, comprising Lactobacillus reuteri NCHBL-005 strain as an active ingredient, and the composition according to the present invention exhibits a treatment or improvement effect on a mouse fibroblast cell line in which a wound is induced.

[0015] The present inventors induced wounds in a mouse fibroblast cell line using a pipette tip and treated it with Lactobacillus rhuteri NCHBL-005 strain, and confirmed that the cell line's migration and proliferation ability was promoted, leading to rapid wound healing. They also confirmed that wound healing was promoted in a protein kinase MAPK-dependent manner.

[0016] Additionally, we confirmed that wounds were induced on the skin of mice and the Lactobacillus rue NCHBL-005 strain was directly applied to the wound site to aid in wound healing.

[0017]

[0018] Hereinafter, the present invention will be described in more detail.

[0019]

[0020] One aspect of the present invention is a Lactobacillus reuteri NCHBL-005 strain deposited under accession number KCTC 15449BP, which has the effect of improving the migration and proliferation of fibroblasts.

[0021]

[0022] Another aspect of the present invention is a pharmaceutical composition for preventing or treating wounds, comprising as an active ingredient Lactobacillus reuteri NCHBL-005 strain, which has the effect of improving the migration and proliferation of fibroblasts.

[0023] In one embodiment of the present invention, the strain may be a Lactobacillus reuteri strain derived from honeybees.

[0024] The strain according to the present invention may be a living cell, a dead cell, or a mixture thereof, and the dead cell may be obtained by killing the strain with heat or radiation, and for example, may be obtained by killing by heat treatment, but is not limited thereto. The strain killed by heat treatment may contain cytoplasm, cell wall, antibacterial substances such as bacteriocin, polysaccharides, organic acids, etc.

[0025] In one embodiment of the present invention, the Lactobacillus reuteri NCHBL-005 strain may be a strain that has been killed by heat at 100°C for 30 minutes.

[0026] The pharmaceutical composition according to the present invention can be used as a pharmaceutical composition comprising a pharmaceutically effective amount of Lactobacillus reuteri NCHBL-005 strain and / or a pharmaceutically acceptable carrier.

[0027] The term "including as an active ingredient" in this specification means including a sufficient amount of Lactobacillus reuteri NCHBL-005 strain in the composition to achieve the desired effect. In this case, the "desired effect" in the present invention may be preventing or treating a wound, and the sufficient amount to achieve such desired effect may be appropriately adjusted depending on the subject of administration, the form of administration, the route of administration, etc.

[0028] The term "pharmaceutically effective amount" in this specification means an amount sufficient to achieve the effect of preventing or treating wounds by enhancing the migration and proliferation of fibroblasts of the Lactobacillus reuteri NCHBL-005 strain described above.

[0029] Pharmaceutically acceptable carriers included in the pharmaceutical composition according to the present invention are those commonly used in formulation, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0030] The pharmaceutical composition of the present invention may further include, in addition to the above ingredients, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc.

[0031] The pharmaceutical composition according to the present invention can be administered to mammals, including humans, via various routes. Any commonly used route of administration may be used, including oral, transdermal, intravenous, intramuscular, and subcutaneous administration.

[0032] The appropriate dosage of the pharmaceutical composition according to the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a generally skilled physician can easily determine and prescribe a dosage effective for the desired treatment or prevention.

[0033] The pharmaceutical composition according to the present invention can be manufactured in a unit dose form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains.

[0034] In the present invention, the Lactobacillus reuteri NCHBL-005 strain may include a 16S rRNA base sequence represented by sequence number 1.

[0035] In the present invention, the Lactobacillus reuteri NCHBL-005 strain may be deposited under the accession number KCTC 15449BP.

[0036] In the present invention, the wound may be at least one selected from the group consisting of burns, thermal injuries, epidermal wounds, ulcers, trauma, post-surgical, childbirth, chronic wounds, and damage caused by dermatitis, but is not limited thereto.

[0037] In the present invention, the pharmaceutical composition may be in one or more formulations selected from the group consisting of liquid, ointment, cream, spray, patch, gel, or aerosol, but is not limited thereto.

[0038] In the present invention, the pharmaceutical composition may enhance the migration and proliferation of fibroblasts in a MAPK (Mitogen-Activated Protein Kinase) dependent manner.

[0039] The term "MAPK (Mitogen-Activated Protein Kinase)" in this specification is well known as a representative signal transduction pathway that transmits extracellular stimuli from the cell membrane to the nucleus within the cell. MAPK transmits signals activated from receptors such as growth hormones, cytokines, and stresses into the cell, and is responsible for various functions such as cell proliferation and differentiation. MAPK can be broadly classified into three types: 1) p38 MAPK, 2) ERK (Extracellular signal-Regulated Kinase), and 3) JNK (c-Jun N-terminal Kinase). ERK plays a central role in cell proliferation and differentiation, and p38 MAPK and JNK are activated by stressful stimuli outside the cell and can mediate inflammatory responses, immune responses, etc.

[0040] In the present invention, the pharmaceutical composition may activate at least one selected from the group consisting of p38 MAPK, ERK (Extracellular signal-Regulated Kinase), and JNK (c-Jun N-terminal Kinase) among the MAPKs.

[0041] In one embodiment of the present invention, the Lactobacillus reuteri NCHBL-005 strain can activate p38 MAPK, ERK, and JNK in mouse fibroblasts to enhance cell migration and proliferation.

[0042]

[0043] Another aspect of the present invention is a cosmetic composition for promoting skin regeneration, comprising as an active ingredient Lactobacillus reuteri NCHBL-005 strain, which has the effect of improving the migration and proliferation of fibroblasts.

[0044] The strain according to the present invention may be a living cell, a dead cell, or a mixture thereof, and the dead cell may be obtained by killing the strain with heat or radiation, and for example, may be obtained by killing by heat treatment, but is not limited thereto. The strain killed by heat treatment may contain cytoplasm, cell wall, antibacterial substances such as bacteriocin, polysaccharides, organic acids, etc.

[0045] The cosmetic composition according to the present invention can be manufactured in any formulation commonly manufactured in the art, and examples thereof include creams, emulsions, toners, packs, foundations, etc. Specifically, the cosmetic composition of the present invention includes formulations of skin lotions, skin softeners, skin toners, astringents, lotions, milk lotions, moisture lotions, nutritional lotions, massage creams, nutritional creams, moisture creams, hand creams, foundations, essences, nutritional essences, packs, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions, and body cleansers.

[0046] In the present invention, the Lactobacillus reuteri NCHBL-005 strain may include a 16S rRNA base sequence represented by sequence number 1.

[0047] In the present invention, the Lactobacillus reuteri NCHBL-005 strain may be deposited under the accession number KCTC 15449BP.

[0048] In the present invention, the cosmetic composition may enhance the migration and proliferation of fibroblasts in a MAPK (Mitogen-Activated Protein Kinase) dependent manner.

[0049] In the present invention, the cosmetic composition may activate at least one selected from the group consisting of p38 MAPK, ERK (Extracellular signal-Regulated Kinase), and JNK (c-Jun N-terminal Kinase) among the MAPKs.

[0050] In one embodiment of the present invention, the Lactobacillus reuteri NCHBL-005 strain can activate p38 MAPK, ERK, and JNK in mouse fibroblasts to enhance cell migration and proliferation.

[0051]

[0052] Another aspect of the present invention is a health functional food composition for promoting skin regeneration, comprising as an active ingredient Lactobacillus reuteri NCHBL-005 strain, which has the effect of improving the migration and proliferation of fibroblasts.

[0053] The strain according to the present invention may be a living cell, a dead cell, or a mixture thereof, and the dead cell may be obtained by killing the strain with heat or radiation, and for example, may be obtained by killing by heat treatment, but is not limited thereto. The strain killed by heat treatment may contain cytoplasm, cell wall, antibacterial substances such as bacteriocin, polysaccharides, organic acids, etc.

[0054] When using the health functional food composition according to the present invention as a food additive, the health functional food composition may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. Generally, when manufacturing a food or beverage, the health functional food composition of the present invention may be added in an amount of 15% by weight or less, preferably 10% by weight or less, relative to the raw materials.

[0055] There are no specific restrictions on the types of the above foods. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and all foods in the conventional sense are included.

[0056] The above beverage may contain various flavoring agents or natural carbohydrates as additional ingredients. The above-mentioned natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, or synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrates may be appropriately determined by those skilled in the art.

[0057] In addition to the above, the health functional food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the health functional food composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit drinks, and vegetable drinks. These ingredients may be used independently or in combination. The proportions of these additives may also be appropriately selected by those skilled in the art.

[0058] The above health functional food can be selected from the group consisting of capsules, tablets, powders, granules, liquids, pills, syrups, and bars.

[0059] In the present invention, the Lactobacillus reuteri NCHBL-005 strain may include a 16S rRNA base sequence represented by sequence number 1.

[0060] In the present invention, the Lactobacillus reuteri NCHBL-005 strain may be deposited under the accession number KCTC 15449BP.

[0061] In the present invention, the health functional food composition may enhance the migration and proliferation of fibroblasts in a MAPK (Mitogen-Activated Protein Kinase) dependent manner.

[0062] In the present invention, the health functional food composition may activate at least one selected from the group consisting of p38 MAPK, ERK (Extracellular signal-Regulated Kinase), and JNK (c-Jun N-terminal Kinase) among the MAPKs.

[0063] In one embodiment of the present invention, the Lactobacillus reuteri NCHBL-005 strain can activate p38 MAPK, ERK, and JNK in mouse fibroblasts to enhance cell migration and proliferation.

[0064] The present invention was conducted to verify the effect of the Lactobacillus reuteri NCHBL-005 strain on wound healing by wounding L929 cell lines (mouse fibroblasts) using a pipette tip and then treating them with the Lactobacillus reuteri NCHBL-005 strain to verify the wound healing effect. As a result, it was confirmed that the wound healing effect was exhibited through the activation of ERK (Extracellular signal-Regulated Kinase), p38 MAPK, and JNK (c-Jun N-terminal Kinase), which are types of MAPK (Mitogen-Activated Protein Kinase), a protein kinase involved in cell proliferation and differentiation.

[0065] Furthermore, after injecting wounds into mouse skin using a punch biopsy device, direct treatment with the Lactobacillus reuteri NCHBL-005 strain was confirmed to be effective in promoting wound healing. Therefore, it can be effectively utilized for wound prevention or treatment and skin regeneration promotion.

[0066] FIG. 1 is a drawing and graph showing an evaluation of the degree of wound healing by treating three types of lactic acid bacteria, including Lactobacillus reuteri NCHBL-005 strain, after wounding L929 cells (mouse fibroblasts) using a pipette tip according to one embodiment of the present invention.

[0067] FIG. 2 shows the results of Western blotting to determine the phosphorylation levels of p38 MAPK, JNK, and ERK after wounding L929 cells (mouse fibroblasts) using a pipette tip and treating them with three types of lactic acid bacteria, including Lactobacillus reuteri NCHBL-005 strain, according to one embodiment of the present invention.

[0068] FIG. 3 is a drawing and graph showing the evaluation of the degree of wound healing by treating L929 cells (mouse fibroblasts) with SB203580 (p38 MAPK inhibitor), SP600125 (JNK inhibitor), and PD0325901 (ERK inhibitor) and then treating them with Lactobacillus reuteri NCHBL-005 strain after making a wound using a pipette tip according to one embodiment of the present invention.

[0069] FIG. 4 is a photograph and graph showing an evaluation of the degree of wound recovery after inducing a wound on a mouse or the like and directly treating the wound site with Lactobacillus reuteri NCHBL-005 strain, according to one embodiment of the present invention.

[0070] A pharmaceutical composition for preventing or treating wounds, comprising as an active ingredient Lactobacillus reuteri NCHBL-005 strain, which has the effect of improving the migration and proliferation of fibroblasts.

[0071] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings so that those skilled in the art can easily practice the present invention. Unless otherwise defined, the terms used in this specification should be interpreted as generally understood by those skilled in the art. The drawings and embodiments of this specification are intended to enable those skilled in the art to easily understand and practice the present invention. Content that may obscure the gist of the invention may be omitted in the drawings and embodiments, and the present invention is not limited to the drawings and embodiments.

[0072] Throughout this specification, "%" used to indicate the concentration of a particular substance is (wt / wt)% for solid / solid, (wt / vol)% for solid / liquid, and (vol / vol)% for liquid / liquid, unless otherwise stated.

[0073]

[0074] Example 1: Isolation and identification of Lactobacillus ruteri NCHBL-005 strain

[0075] To isolate lactic acid bacteria from the gut microbiota of honeybees, the target insects were first collected in the Gwangju area, placed in sterilized collection containers, and refrigerated for 10 minutes for each individual. The gastrointestinal tracts were aseptically removed using microsurgical instruments on a green bench. The honeybee gut microbiota was obtained by suspending the extracted gastrointestinal contents in 10 ml of sterile Tween 80-added 0.1% peptone physiological saline (0.9% w / v NaCl, 0.1% w / v Tween 80, 0.1% w / v peptone).

[0076] Lactic acid bacteria were cultured in a liquid medium (Difco) containing 500 ㎕ of a suspension of the intestines. TM After serially diluting appropriately using Lactobacilli MRS Broth, BD, USA), the solid medium (BBL) was grown using a platinum sieve. TM Spread on LBS Agar (BD, USA) and BD GasPak TM EZdp was added and cultured at 37℃ until bacterial colonies were confirmed. Bacterial colonies confirmed on LBS solid medium were identified as Lactobacillus based on the Gram-positive result of Gram stain and the catalase-negative reaction confirmed when mixed with 3% hydrogen peroxide. The isolated field isolate Lactobacillus was stored at -20℃ in MRS medium containing 15% glycerol and used in the following experiments.

[0077] Phylogenetic identification of Lactobacillus reuteri NCHBL-005 among the isolated strains was performed through base sequence analysis of the 16S rRNA gene, and the 16S rRNA gene of Lactobacillus strains was compared based on the similarity of the primary and secondary structures of the 16S rRNA gene using the PHYDIT program. The 16S rRNA base sequence of Lactobacillus reuteri NCHBL-005 is shown in Table 1 below.

[0078] 서열번호명칭서열1LactobacillusreuteriNCHBL-00516S rRNACTGATTGATGGTGCTTGCACCTGATTGACGATGGATCACCAGTGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAACAACAAAAGCCACATGGCTTTTGTTTGAAAGATGGCTTTGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCGTGAGAGTAACTGTTCACGCAGTGACGGTATCCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGGTTGAAACTCAA

[0079]

[0080] Example 2: Cultivation of Lactobacillus rhuteri NCHBL-005 strain

[0081] Lactobacillus reuteri NCHBL-005 strain was streaked onto MRS agar, a selective medium for lactic acid bacteria, and cultured in an incubator at 37°C for 24 hours to allow single colonies to grow. Afterwards, the single colonies were transferred to MRS liquid medium using a sterilized tip and sufficiently pre-cultured for 12 to 16 hours.

[0082] Afterwards, to obtain highly active bacteria, a small amount of the pre-cultured bacteria was transferred to a new MRS liquid medium and cultured (10-fold dilution). Afterwards, the bacteria were harvested at an optical density (OD) of 0.6, which is the point at which the bacteria were most active, through absorbance measurement. The lactic acid bacteria were centrifuged, washed with PBS, and then killed by heat (100°C, 30 minutes). The killed cells were then freeze-dried and used.

[0083]

[0084] Example 3: Experimental evaluation of the wound healing ability of Lactobacillus rhuteri NCHBL-005 strain using L929 cells.

[0085] To evaluate the wound healing ability of Lactobacillus reuteri NCHBL-005 strain, a mouse-derived fibroblast cell line, L929 (Korea Cell Line Bank, KCLB No. 10001 NCTC clone 929), was used. L929 cells were seeded on a plate, and after 24 hours, the center of the cell line was scratched using a pipette tip to create a wound. Then, the lactic acid bacteria strains (Lactobacillus plantarum NCHBL-004, Lactobacillus kunkeei NCHBL-003, Lactobacillus reuteri NCHBL-005) were treated at 50 μg / ml. The change in wound size (%) after strain treatment for 72 hours is presented in Fig. 1 and Table 2 below.

[0086] Change in wound size (%) Control L. plantaru mL. kunkeei L. reuteri 0 h100 100 100 100 24 h90 61 50 389 0. 82 19 65 339 1. 03 28 72 67 84 78 36 86 67 48 h84 90 51 66 338 0. 48 38 12 80 48 79 46 67 59 55 39 31 72 h74 18 80 77 335 6. 46 03 09 67 65 59 67 19 67 25 57 27 44

[0087] The experimental results showed that even among the same Lactobacillus strains, the Lactobacillus reuteri NCHBL-005 strain promoted the migration and proliferation of L929 cells and most effectively reduced the wound size (%) compared to other Lactobacillus strains. These results demonstrate that the Lactobacillus reuteri NCHBL-005 strain can be effectively used for wound treatment by influencing the migration and proliferation of mouse-derived fibroblasts in vitro.

[0088]

[0089] Example 4: Experiment to confirm the wound healing effect of Lactobacillus rhubarb NCHBL-005 strain by phosphorylation of MAPK in L929 cells.

[0090] To evaluate the degree of activation of MAPK, a protein kinase involved in cell migration and proliferation, lactic acid bacteria strains (Lactobacillus plantarum NCHBL-004, Latobacillus kunkeei NCHBL-003, Lactobacillus reuteri NCHBL-005) were treated at 50 ㎍ / ㎖ to wounded mouse-derived L929 cells, and then the cells were disrupted at 0, 30, and 60 minutes to extract proteins.

[0091] To evaluate the phosphorylation level of p38 MAPK, JNK, and ERK, which are types of MAPK,

[0092] anti-pp38 (cell signaling technology, cat. 9211s);

[0093] anti-pJNK (cell signaling technology, cat. 9251s);

[0094] anti-pERK (santa cruz, cat. Sc-7383); and

[0095] The degree of phosphorylation of each protein was measured using anti-actin antibody (Santa Cruz, cat. sc-47778).

[0096] As a result of the measurement, as shown in Fig. 2, it was confirmed through Western blotting that the Lactobacillus reuteri NCHBL-005 strain promotes the phosphorylation of protein kinases (p38 MAPK, JNK, ERK) involved in the migration and proliferation of L929 cells more than other Lactobacillus strains, even though they are the same Lactobacillus strains. Specifically, when the Lactobacillus reuteri NCHBL-005 strain was treated, significantly more phosphorylated p38 MAPK, JUK, and ERK were detected than when the other Lactobacillus strains were treated.

[0097]

[0098] Example 5: Experiment to confirm the inhibition of the wound healing effect of Lactobacillus ruteri NCHBL-005 strain using a MAPK inhibitor in L929 cells.

[0099] To determine whether Lactobacillus reuteri NCHBL-005 strain exhibits a wound healing effect dependent on MAPK, a protein kinase involved in cell migration and proliferation, a MAPK inhibitor was used.

[0100] SB203580 inhibits the activation of p38 MAPK;

[0101] SP600125 inhibits the activation of JNK;

[0102] Additionally, PD0325901 is known to inhibit the activation of ERK.

[0103] To evaluate the degree of wound healing of Lactobacillus reuteri NCHBL-005 strain when each protein kinase was inhibited, the inhibitors SB203580 (Selleckchem, cat. S1076), SP600125 (Selleckchem, cat. S1460), and PD032901 (Selleckchem, cat. S1036) and Lactobacillus reuteri NCHBL-005 strain were simultaneously treated at a concentration of 20 μM to wounded mouse-derived L929 cells. The change in wound size (%) for 72 hours after treatment is presented in Fig. 3 and Table 3 below.

[0104] Control(%)SB203580Treatment(%)SP600125Treatment(%)PD0325901Treatment(%)L.reuteri(%)0 h10010010010010024 h93.5379966793.7022492.7386866790.9785033388.6081233348 h80.8890533380.9602933386.6889866777.1149633365.4215966772 h65.5041133374.0553033382.7074856.9744434.40421333

[0105] Experimental results showed that wound healing was inhibited when each inhibitor was administered, compared to the group administered Lactobacillus reuteri NCHBL-005 strain alone. These results confirmed that Lactobacillus reuteri NCHBL-005 strain affects the migration and proliferation of mouse-derived fibroblasts in a MAPK-dependent manner in vitro, specifically through the activation of p38 MAPK, JNK, and ERK.

[0106]

[0107] Example 6: Experiment to confirm the wound healing effect of Lactobacillus rhubari NCHBL-005 strain as an external agent.

[0108] To determine whether Lactobacillus reuteri NCHBL-005 strain has a wound healing effect as an external agent, the skin of Balb / c mice was wounded using a skin punch biopsy ([Kai-medical] Disporsable Biopsy Punch, cat. BP-60F), and 10 μl of Lactobacillus reuteri NCHBL-005 strain at 10 μg / ml was directly applied to the wound. The change in wound size (%) for 10 days after application is presented in Fig. 4 and Table 4 below.

[0109] DayControl(%)L.reuteri10 ㎍ / ㎖(%)0100100299.2765882.45465478.7216960.18968651.2508226.23795830.207126.7742461017.960380

[0110] The experimental results confirmed that the group treated with the Lactobacillus reuteri NCHBL-005 strain accelerated wound healing compared to the untreated group. Specifically, the wound healed within 10 days after treatment with the Lactobacillus reuteri NCHBL-005 strain. These results confirmed that directly applying the Lactobacillus reuteri NCHBL-005 strain to skin wounds can also aid wound healing.

[0111]

[0112] [Accession number]

[0113] Name of depositor: Korea Research Institute of Bioscience and Biotechnology

[0114] Accession number: KCTC 15449BP

[0115] Date of acceptance: 20230519

[0116] The present invention relates to a pharmaceutical composition for preventing or treating wounds, comprising as an active ingredient Lactobacillus reuteri NCHBL-005 strain, which has the effect of improving the migration and proliferation of fibroblasts.

[0117]

[0118] [Deposit Certificate]

[0119]

Claims

1. Lactobacillus reuteri NCHBL-005 strain, which has the effect of improving the migration and proliferation of fibroblasts.

2. A pharmaceutical composition for preventing or treating wounds, comprising as an active ingredient Lactobacillus reuteri NCHBL-005 strain, which has the effect of improving the migration and proliferation of fibroblasts.

3. A pharmaceutical composition for preventing or treating a wound, wherein the Lactobacillus reuteri NCHBL-005 strain in the second paragraph comprises a 16S rRNA base sequence represented by sequence number 1.

4. A pharmaceutical composition for preventing or treating wounds, wherein the Lactobacillus reuteri NCHBL-005 strain in paragraph 2 is deposited under the accession number KCTC 15449BP.

5. A pharmaceutical composition for preventing or treating a wound, wherein the wound is at least one selected from the group consisting of burns, thermal injuries, epidermal wounds, ulcers, trauma, post-surgical, childbirth, chronic wounds, and damage caused by dermatitis, according to any one of claims 2 to 4.

6. A pharmaceutical composition for preventing or treating a wound, wherein the pharmaceutical composition is in one or more formulations selected from the group consisting of a liquid, an ointment, a cream, a spray, a patch, a gel, or an aerosol, according to any one of claims 2 to 4.

7. A pharmaceutical composition for preventing or treating wounds, wherein the pharmaceutical composition according to any one of claims 2 to 4 enhances the migration and proliferation of fibroblasts in a MAPK (Mitogen-Activated Protein Kinase) dependent manner.

8. A pharmaceutical composition for preventing or treating a wound, wherein the pharmaceutical composition in claim 7 activates at least one selected from the group consisting of p38 MAPK, ERK (Extracellular signal-regulated Kinase), and JNK (c-Jun N-terminal Kinase) among the MAPKs.

9. A cosmetic composition for promoting skin regeneration, comprising as an active ingredient Lactobacillus reuteri NCHBL-005 strain, which has the effect of improving the migration and proliferation of fibroblasts.

10. A cosmetic composition for promoting skin regeneration, wherein the Lactobacillus reuteri NCHBL-005 strain in paragraph 9 contains a 16S rRNA base sequence represented by sequence number 1.

11. A cosmetic composition for promoting skin regeneration, wherein the Lactobacillus reuteri NCHBL-005 strain in paragraph 9 is deposited under the accession number KCTC 15449BP.

12. A cosmetic composition for promoting skin regeneration, according to any one of claims 9 to 11, wherein the cosmetic composition enhances the migration and proliferation of fibroblasts in a MAPK (Mitogen-Activated Protein Kinase) dependent manner.

13. A cosmetic composition for promoting skin regeneration, wherein the cosmetic composition in claim 12 activates at least one MAPK selected from the group consisting of p38 MAPK, ERK (Extracellular signal-Regulated Kinase), and JNK (c-Jun N-terminal Kinase).

14. A health functional food composition for promoting skin regeneration, comprising as an active ingredient Lactobacillus reuteri NCHBL-005 strain, which has the effect of improving the migration and proliferation of fibroblasts.

15. A health functional food composition for promoting skin regeneration, wherein the Lactobacillus reuteri NCHBL-005 strain in claim 14 comprises a 16S rRNA base sequence represented by sequence number 1.

16. A health functional food composition for promoting skin regeneration, wherein the Lactobacillus reuteri NCHBL-005 strain in paragraph 14 is deposited under the accession number KCTC 15449BP.

17. A health functional food composition for promoting skin regeneration, wherein the health functional food composition according to any one of claims 14 to 16 enhances the migration and proliferation of fibroblasts in a MAPK (Mitogen-Activated Protein Kinase) dependent manner.

18. In claim 17, the health functional food composition is a health functional food composition for promoting skin regeneration that activates at least one selected from the group consisting of p38 MAPK, ERK (Extracellular signal-Regulated Kinase), and JNK (c-Jun N-terminal Kinase) among the MAPKs.

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