Vesicles derived from Lactobacillus paracasei and uses thereof

Abstract

The present invention relates to vesicles derived from Lactobacillus paracasei and uses thereof, and more particularly to a composition for improving, preventing, and treating inflammatory diseases and cancer, which contains vesicles derived from Lactobacillus paracasei as an active ingredient, which can effectively suppress various inflammatory reactions.

Description

[Technical field]

[0001] The present invention relates to vesicles derived from Lactobacillus paracasei and uses thereof, and more particularly to a composition for preventing or treating inflammatory diseases and cancer, which contains vesicles derived from Lactobacillus paracasei as an active ingredient.

[0002] This application claims priority to Korean Patent Application No. 10-2018-0174027 filed on December 31, 2018, and Korean Patent Application No. 10-2019-0048671 filed on April 25, 2019, the entire contents of which are incorporated herein by reference in their entirety in the specification and drawings. [Background technology]

[0003] In the 21st century, the importance of acute infectious diseases, which were once considered to be infectious, has decreased, while disease patterns have changed to chronic diseases that reduce quality of life and are major diseases that determine human lifespan. The aforementioned chronic diseases are characterized by chronic inflammation accompanied by immune dysfunction, and various chronic inflammatory diseases and cancers that arise as a result of this have become major issues for public health.

[0004] Inflammation is a local or systemic defense mechanism against cell and tissue damage or infection, and is mainly induced by a chain of biological reactions caused by direct reactions of humoral mediators that constitute the immune system or stimulation of local or systemic effector systems. Major inflammatory diseases include gastrointestinal diseases such as gastritis and inflammatory bowel disease, oral diseases such as periodontitis, respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and rhinitis, skin diseases such as atopic dermatitis, alopecia, and psoriasis, arthritis such as degenerative arthritis and rheumatoid arthritis, and cancer.

[0005] The general compositions used for treating or preventing chronic inflammatory diseases are largely classified into steroidal and non-steroidal compositions, and most of them often have various side effects. Therefore, interest in TNF-α inhibitors, which are proinflammatory cytokines known to be the main cause of chronic inflammatory diseases, has recently been increasing.

[0006] However, at present, there is a lack of development of drugs that have low side effects and can be effectively used to prevent and treat chronic inflammatory diseases by effectively suppressing the expression of TNF-α, and the same is true for cancer therapeutic agents. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been devised to solve the problems in the prior art as described above, and aims to provide a composition for improving, preventing or treating inflammatory diseases and cancer, which contains vesicles derived from Lactobacillus paracasei as an active ingredient.

[0008] However, the technical problems that the present invention aims to achieve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those having ordinary skill in the art to which the present invention pertains from the following description. [Means for solving the problem]

[0009] The present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases or cancer, comprising vesicles derived from Lactobacillus paracasei as an active ingredient.

[0010] The present invention also provides a food composition for preventing or ameliorating inflammatory diseases or cancer, which contains vesicles derived from Lactobacillus paracasei as an active ingredient.

[0011] The present invention also provides an inhalant composition for preventing or treating inflammatory diseases or cancer, which comprises vesicles derived from Lactobacillus paracasei as an active ingredient.

[0012] The present invention also provides a cosmetic composition for preventing or ameliorating inflammatory diseases, which comprises vesicles derived from Lactobacillus paracasei as an active ingredient.

[0013] The inflammatory disease of the present invention is a disease mediated by TNF-α or IL-1β, and includes inflammatory enteritis (acute enteritis, chronic enteritis, etc.), gastritis, asthma, chronic obstructive pulmonary disease (COPD), rhinitis, atopic dermatitis, alopecia, psoriasis, degenerative arthritis, and rheumatoid arthritis, but is not limited thereto as long as it is a disease caused by an inflammatory response.

[0014] The cancers of the present invention include, but are not limited to, colon cancer, gastric cancer, lung cancer, liver cancer, biliary tract cancer, pancreatic cancer, breast cancer, ovarian cancer, kidney cancer, bladder cancer, and prostate cancer.

[0015] In one embodiment of the present invention, the vesicles preferably have an average diameter of 10 to 300 nm, but are not limited thereto as long as they are vesicles naturally or artificially secreted or isolated from Lactobacillus paracasei.

[0016] In another embodiment of the present invention, the vesicles can be isolated from a culture medium of Lactobacillus paracasei or a food cultured with Lactobacillus paracasei, but is not limited thereto.

[0017] The present invention also provides a method for treating an inflammatory disease or cancer, comprising the step of administering to an individual a composition containing vesicles derived from Lactobacillus paracasei as an active ingredient.

[0018] The present invention also provides a use of a composition containing vesicles derived from Lactobacillus paracasei as an active ingredient for the prevention and / or treatment of inflammatory diseases or cancer.

[0019] The present invention also provides a use of vesicles derived from Lactobacillus paracasei for producing a medicament for use in treating inflammatory diseases and / or cancer. Effect of the Invention

[0020] The vesicles derived from Lactobacillus paracasei according to the present invention can effectively suppress the expression of proinflammatory cytokines such as TNF-α and IL-1β, which are known to be factors in various chronic inflammatory diseases, and are therefore expected to be widely used as an agent for improving, preventing, or treating various chronic inflammatory diseases and cancers with low side effects. [Brief description of the drawings]

[0021] [Figure 1] 1a and 1b are diagrams showing the analysis results of vesicles derived from Lactobacillus paracasei according to one embodiment of the present invention. [Diagram 2] 2a and 2b are diagrams showing the results of confirming the cytotoxicity and proinflammatory effects of vesicles derived from Lactobacillus paracasei according to one embodiment of the present invention. [Diagram 3] FIG. 3 is a diagram showing the results of confirming the anti-inflammatory effect of vesicles derived from Lactobacillus paracasei according to one embodiment of the present invention. [Figure 4] 4a and 4b are diagrams showing the results of qRT-PCR confirming the effect of Lactobacillus paracasei-derived vesicles according to one embodiment of the present invention on inhibiting LPS-induced inflammatory response. [Diagram 5] 5a and 5b are diagrams showing the results of Western blotting to confirm the effect of Lactobacillus paracasei-derived vesicles according to one embodiment of the present invention on inhibiting LPS-induced inflammatory response. [Figure 6]6a to 6e are diagrams showing the results of histological analysis of the inflammatory response suppressing effect of Lactobacillus paracasei-derived vesicles in DSS-induced colitis according to one embodiment of the present invention. [Figure 7] 7a to 7d show the results of qRT-PCR confirmation of the inflammatory response suppressing effect of Lactobacillus paracasei-derived vesicles in DSS-induced colitis according to one embodiment of the present invention. [Figure 8] 8a and 8b show the results of Western blotting to confirm the inflammatory response suppressing effect of Lactobacillus paracasei-derived vesicles in DSS-induced colitis according to one embodiment of the present invention. [Figure 9] FIG. 9 is a graph showing the results of measuring the size of cancer over time after oral administration of vesicles derived from Lactobacillus paracasei in a cancer model prepared by transplanting cancer cells according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] In the present invention, it was confirmed that vesicles derived from Lactobacillus paracasei effectively suppress the expression of various factors related to inflammatory responses and exhibit a remarkable therapeutic effect on inflammatory diseases. Therefore, it is expected that the vesicles derived from Lactobacillus paracasei of the present invention can be used in various ways to improve, prevent, and treat various chronic inflammatory diseases and cancers.

[0023] In the present specification, the term "extracellular vesicle" refers to a structure consisting of a nano-sized membrane secreted from various bacteria, and in the present invention, it collectively refers to all structures consisting of a membrane naturally secreted from Lactobacillus paracasei or artificially produced. The vesicles can be separated from a culture solution containing Lactobacillus paracasei cells by one or more methods selected from the group consisting of centrifugation, ultra-high speed centrifugation, high pressure treatment, extrusion, ultrasonic decomposition, cell lysis, homogenization, freezing-thawing, electroporation, mechanical decomposition, chemical treatment, filtration through a filter, gel filtration chromatography, free-flow electrophoresis, and capillary electrophoresis. In addition, the process may further include washing for removing impurities and concentrating the collected vesicles.

[0024] In this specification, the term "inflammatory disease" refers to a disease induced by an inflammatory response in the body, and representative examples include respiratory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and rhinitis; skin inflammatory diseases such as atopic dermatitis, psoriasis, acne, contact dermatitis, and alopecia; digestive inflammatory diseases such as gastritis, digestive ulcer, and inflammatory bowel disease (acute enteritis, Crohn's disease, inflammatory bowel disease, Behcet's colitis, ulcerative colitis, bacterial colitis, etc.); vaginitis; arthritis such as osteoarthritis and rheumatoid arthritis; and complications thereof. The inflammatory disease is used to mean not only general inflammatory diseases but also metabolic diseases associated with inflammatory responses, and includes, for example, diabetes, obesity, hepatitis, and cirrhosis of the liver.

[0025] As used herein, "prevention" refers to any action of suppressing or delaying the onset of inflammatory diseases and cancer by administering the food, cosmetic, inhalant, or pharmaceutical composition according to the present invention.

[0026] As used herein, "treatment" refers to any action that improves or favorably alters the symptoms of inflammatory diseases and cancer by administering a food, cosmetic, inhalant, or pharmaceutical composition according to the present invention.

[0027] As used herein, "improvement" refers to any action that at least reduces a parameter associated with the condition being treated, eg, the severity of a symptom.

[0028] As used herein, "individual" refers to a subject in need of treatment for a disease, and more specifically refers to mammals, such as humans or non-human primates, mice, rats, dogs, cats, horses, and cows.

[0029] In the present specification, the term "pharmaceutical composition" refers to a pharmaceutical composition in the form of a capsule, tablet, granule, injection, ointment, powder, or drink, and the pharmaceutical composition is intended for humans. The pharmaceutical composition may be formulated and used in the form of an oral dosage form such as a powder, granule, capsule, tablet, aqueous suspension, external preparation, suppository, or sterile injection solution by a conventional method, but is not limited thereto. The pharmaceutical composition of the present invention may contain a pharmaceutical acceptable carrier. The pharmaceutical acceptable carrier may be a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a dye, a flavoring, etc., when administered orally, and may be used in the form of a mixture of a buffer, a preservative, a soothing agent, a solubilizer, an isotonic agent, a stabilizer, etc., when administered locally. A base, an excipient, a lubricant, a preservative, etc., may be used. The pharmaceutical composition of the present invention may be prepared in various dosage forms by mixing with the above-mentioned pharma- ceutical acceptable carriers. For example, for oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it may be prepared in unit dose ampoules or multiple dose forms. In addition, it may be prepared in the form of solutions, suspensions, tablets, capsules, sustained release preparations, etc.

[0030] On the other hand, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, etc. Also, fillers, anti-agglomerating agents, lubricants, wetting agents, flavors, emulsifiers, preservatives, etc. may be additionally included.

[0031] The routes of administration of the pharmaceutical compositions according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, percutaneous, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal. Oral or parenteral administration is preferred. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention may also be administered in the form of suppositories for rectal administration.

[0032] The pharmaceutical composition of the present invention may vary depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route, and period, and may be appropriately selected by those skilled in the art and may be administered at 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. The dosage may be administered once a day or in several divided doses. The dosage does not limit the scope of the present invention in any aspect. The pharmaceutical composition according to the present invention may be formulated as a pill, sugar-coated tablet, capsule, liquid, gel, syrup, slurry, or suspension.

[0033] In the present invention, the food composition can be used in various foods, such as beverages, gums, teas, vitamin complexes, dietary supplements, etc., and can be in the form of pills, powders, granules, infusions, tablets, capsules, or beverages. The food composition includes a functional health food composition. In this case, the amount of the vesicles derived from Lactobacillus paracasei in the food or beverage can be generally added at 0.01 to 15% by weight of the total food weight in the case of the food composition of the present invention, and at 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 mL in the case of the health drink composition. In this specification, functional health food refers to a food manufactured and / or processed using raw materials or ingredients that have functions useful to the human body, and has characteristics in its ingredients compared to general foods, and has a more active health maintenance, or a desired or expected effect.

[0034] The food composition of the present invention may contain conventional food additives in the art, such as flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, etc. The food composition of the present invention is not particularly limited to the components added other than the vesicles derived from Lactobacillus paracasei as an essential component, and may contain various flavoring agents or natural carbohydrates, etc. as additional components, like conventional foods. Examples of the natural carbohydrates include conventional sugars such as monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; polysaccharides, e.g., dextrin, cyclodextrin; and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 mL of the composition of the present invention.

[0035] In addition to the above, the food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavorings such as synthetic flavorings and natural flavorings, coloring agents and enhancers (cheese, chocolate, etc.), pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc. Such ingredients may be used independently or in combination. The ratio of such additives is not particularly important, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

[0036] In the present invention, the cosmetic composition may contain 0.0005-50% by weight of the vesicles derived from Lactobacillus paracasei based on the total weight of the composition. The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions as the vesicles derived from Lactobacillus paracasei.

[0037] The cosmetic product prepared from the cosmetic composition of the present invention can be prepared in the form of a general emulsion formulation and a general solubilization formulation. The emulsion formulation includes nutritional lotions, creams, essences, etc., and the solubilization formulation includes softening lotions. In addition, by containing a dermatologically acceptable medium or base, the cosmetic product can be prepared in the form of a topical or systemic application auxiliary commonly used in the dermatological field.

[0038] Suitable cosmetic formulations may be, for example, in the form of solutions, gels, solids or pastes of anhydrous products, emulsions obtained by dispersing an oily phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes), non-ionic vesicular dispersions, creams, skins, lotions, powders, ointments, sprays or conceal sticks, or may be produced in the form of foams or aerosol compositions further containing a compressed propellant.

[0039] The cosmetic composition of the present invention may also contain additional adjuvants commonly used in the cosmetic or dermatological fields, such as fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizing agents, foaming agents, fragrances, surfactants, water, ionic or non-ionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, moisturizing agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredient commonly used in cosmetics, and said ingredients may be introduced in amounts commonly used in the dermatological field.

[0040] Examples of products to which the cosmetic composition of the present invention can be added include cosmetics such as astringent lotions, softening lotions, nourishing lotions, various creams, essences, packs, foundations, etc., as well as cleansers, facial washes, soaps, treatments, beauty serums, etc.

[0041] Specific formulations of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, essence, nourishing essence, pack, soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, emulsion, pressed powder, loose powder, eye shadow, and the like.

[0042] In the inhalant composition of the present invention, the active ingredient may be added directly to the inhalant or may be used together with other ingredients, and may be used appropriately by a conventional method. The amount of the active ingredient to be mixed may be determined to suit the purpose of use (prophylaxis or treatment).

[0043] The present invention also provides a method for treating an inflammatory disease or cancer, comprising the step of administering to an individual a composition containing vesicles derived from Lactobacillus paracasei as an active ingredient.

[0044] The present invention also provides a use of a composition containing vesicles derived from Lactobacillus paracasei as an active ingredient for the prevention and / or treatment of inflammatory diseases or cancer.

[0045] The present invention also provides a use of vesicles derived from Lactobacillus paracasei for producing a medicament for inflammatory diseases or cancer.

[0046] In the present invention, "administration" means introducing the pharmaceutical composition of the present invention into a patient by any suitable method, and the administration route of the composition of the present invention may be through various routes, such as oral or parenteral, as long as it can reach the target tissue.

[0047] By "individual" in the present invention is meant a subject in need of treatment for a disease, and more specifically means a mammal, such as a human or non-human primate, mouse, dog, cat, horse, or cow.

[0048] In the following, preferred examples are presented to aid in understanding the present invention. However, the following examples are merely provided to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples. EXAMPLES

[0049] [Example 1: Isolation of vesicles derived from Lactobacillus paracasei] To isolate extracellular vesicles (EVs) from Lactobacillus paracasei, Lactobacillus paracasei was inoculated into MRS (de Man-Rogosa and Sharpe) medium and measured by optical density (OD) at 200 rpm at 37 °C. 600nm) to 1.0-1.5, and then reinoculated into Luria Bertani (LB) medium and cultured. The culture medium containing the bacteria was then collected and centrifuged at 10,000g for 20 minutes at 4°C to obtain a supernatant from which the bacteria had been removed. The obtained supernatant was further filtered using a 0.22 μm filter, and the filtered supernatant was concentrated to a volume of 50 mL or less using a 100 kDa Pellicon 2 Cassette filter membrane (Merck Millipore) and a MasterFlex pump system (Cole-Parmer). The concentrated supernatant was further filtered using a 0.22 μm filter to separate vesicles derived from Lactobacillus paracasei. The amount of protein in the supernatant was measured using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific). The isolated vesicles were then observed using a JEM1011 transmission electron microscope (TEM, JEOL) and a Zetasizer Nano ZS (Malvern Instruments). As a control, vesicles derived from E. coli were isolated in the same manner. The results are shown in Figure 1a and Figure 1b.

[0050] As shown in Figures 1a and 1b, the average diameter of the vesicles isolated from the culture medium of Lactobacillus paracasei was 34.22 nm, confirming that the vesicles were successfully isolated.

[0051] [Example 2: Preparation of Lactobacillus paracasei cells] To compare the vesicles and cells derived from Lactobacillus paracasei, Lactobacillus paracasei cells were prepared. More specifically, Lactobacillus paracasei cells cultured in the same manner as in Example 1 were heat-treated at 70°C for 1 hour, then centrifuged at 10,000g at 4°C for 20 minutes to obtain heat-inactivated cells from which the supernatant was removed. The cells were then resuspended in phosphate buffered saline (PBS), and the protein content was measured using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific).

[0052] [Example 3: Confirmation of the inflammatory effect of vesicles derived from Lactobacillus paracasei] To confirm the effect of Lactobacillus paracasei vesicles on inflammatory responses in inflammatory cells, mouse macrophage strain Raw264.7 was treated with Lactobacillus paracasei vesicles at concentrations of 0.1, 1, and 10 μg / mL, and the degree of cell death and the amount of TNF-α secreted were measured after reaction. More specifically, 5 × 10 Raw264.7 cells were dispensed into a 48-well plate, treated with Lactobacillus paracasei vesicles diluted in DMEM serum-free medium, and cultured for 12 hours. The degree of cell death was measured using EZ-CYTOX (Dogen), and the cell culture medium was placed in a 1.5 mL tube, centrifuged at 3,000 g for 5 minutes, and the supernatant was collected and stored at -80 °C, after which ELISA was performed. For ELISA, the capture antibody (Abcam) was diluted in phosphate buffer solution and dispensed into a 96-well plate at 50μL each, and then reacted at 4℃ for 16 hours. After washing three times with 100μL of phosphate buffer solution (PBST) supplemented with 0.05% Tween 20, 100μL of phosphate buffer solution (RD) supplemented with 1% BSA (bovine serum albumin) was added per well and blocked at room temperature for 1 hour. Then, 50μL of the sample and standard to be tested were added according to the concentration, reacted at room temperature for 2 hours, and washed three times with PBST. Then, the detection antibody was diluted in RD, added at 50μL per well, reacted at room temperature for 2 hours, washed three times with PBST to thoroughly remove unbound antibodies, and 50μL of streptavidin-HRP (R&D system) diluted 1 / 40 in RD was added per well and reacted at room temperature for 20 minutes. Finally, after washing three times with 100 μL of PBST, 50 μL of TMB substrate (SurModics) was added per well, and after 5 to 20 minutes, when color development occurred, 50 μL of 1M sulfuric acid solution was added per well to terminate the reaction, and the absorbance was measured at 450 nm using a SpectraMax M3 microplate reader (Molecular Devices). The results are shown in Figure 2a and Figure 2b.

[0053] As shown in Figure 2a, Lactobacillus paracasei-derived vesicles did not affect cell viability, and as shown in Figure 2b, they promoted the secretion of inflammatory mediators in mouse macrophage strains when treated with high concentrations of 10 μg / mL or more.

[0054] [Example 4: Confirmation of the anti-inflammatory effect of vesicles derived from Lactobacillus paracasei] In order to confirm the anti-inflammatory effect of Lactobacillus paracasei-derived vesicles, Raw264.7, a mouse macrophage strain, was pretreated with Lactobacillus paracasei-derived vesicles at concentrations of 0.1, 1, 10, and 50 μg / mL, and then cultured, and the amount of TNF-α secreted was measured. More specifically, 5×10 Raw264.7 cells were dispensed into a 48-well plate, and then Lactobacillus paracasei cells prepared in the same manner as in Example 2 were treated with Lactobacillus paracasei-derived vesicles diluted with DMEM serum-free medium, and cultured for 12 hours. Then, the cells were further treated with vesicles derived from E. coli at a concentration of 1 μg / mL, and further cultured for 12 hours. Then, the amount of TNF-α secreted was measured in the same manner as in Example 3. The results are shown in FIG. 3.

[0055] As shown in Figure 3, the samples pretreated with Lactobacillus paracasei cells did not significantly reduce the secretion of TNF-α by E. coli-derived vesicles, whereas the samples treated with Lactobacillus paracasei-derived vesicles significantly reduced the secretion of TNF-α by E. coli-derived vesicles depending on the concentration. These results confirmed that inflammatory responses induced by pathogenic bacteria or vesicles derived from pathogenic bacteria can be effectively suppressed using Lactobacillus paracasei-derived vesicles.

[0056] [Example 5: Confirmation of the effect of Lactobacillus paracasei-derived vesicles in suppressing LPS-induced inflammatory response] 5.1.qRT-PCR Experiments To confirm the inhibitory effect of LPS (lipopolysaccharide)-induced inflammatory responses of Lactobacillus paracasei-derived vesicles, HT29 cells, a colorectal cancer cell line, were treated with LPS and / or Lactobacillus paracasei-derived vesicles. More specifically, HT29 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin, and the cells were washed with phosphate buffered saline. After culturing in serum-free DMEM medium for 3 hours, the cells were pretreated with Lactobacillus paracasei-derived vesicles at a concentration of 500 ng / mL and cultured for 12 hours. The cultured cells were treated with 1 μg / mL LPS and cultured for another 12 hours. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was then performed. To perform qRT-PCR, total RNA was extracted from the cultured cells using TRIZOL reagent (Invitrogen) according to the protocol provided by the manufacturer, and the amount of RNA was measured using a NanoDrop 2000 Spectrophotometer. cDNA was synthesized using M-MLV reverse transcription polymerase, and qRT-PCR was performed using the synthesized cDNA as a template. qRT-PCR was performed using Quant3 Studio Real-time PCR Systems (Applied Biosystems), and quantification was performed using the CyberGreen qPCR Mastermix kit according to the protocol provided by the manufacturer. The primer sequences used are listed in Table 1, and the results are shown in Figures 4a and 4b.

[0057] [Table 1]

[0058] As shown in Figure 4a and 4b, LPS promotes inflammatory responses and increases the expression of pro-inflammatory cytokines IL-1α, IL-1β, IL-2, IL-3, and TNF-α, but in cells pretreated with Lactobacillus paracasei-derived vesicles (LpEV), the increased expression of pro-inflammatory cytokines was suppressed.In addition, the expression of IL-10 and TGF-β was increased.

[0059] [5.2. Western blotting experiment] Western blotting was performed to confirm the inhibitory effect of Lactobacillus paracasei-derived vesicles on LPS-induced inflammatory responses. Cells prepared in the same manner as in Example 5.1 were washed twice with cold phosphate buffer and lysed with RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM NaF, 0.1 mM phenylmethanesulfonylfluoride, and 0.5% protease inhibitor cocktail), centrifuged at 12,000Xg for 5 minutes at 4℃ to thoroughly remove cell debris, and the amount of protein was measured using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Then, 40 μg of protein was subjected to SDS-PAGE (Bio-Rad Laboratories) using 8-12% SDS-polyacrylamide gel to separate the protein, and the separated protein was then transferred to Hybond TMProteins were transferred to an ECL nitrocellulose membrane (Amersham Bioscience). The membrane to which the proteins were transferred was blocked at room temperature for 1 hour using TBST supplemented with 5% skim milk, and then treated with a primary antibody diluted 1:2,000 and incubated at 4°C for 16 hours. After washing three times with TBST, the membrane was treated with a secondary antibody conjugated with HRP diluted 1:2,000 and incubated at room temperature for 1 hour, and observed using an ECL detection system (Amersham). Antibodies against COX-2 and activated MMP9 were purchased from SantaCruz, antibodies against NFκ and iNOS were purchased from BioLegend, and antibodies against β-actin, phosphorylated-Iκκ, and Iκ were purchased from Cell Signaling Technology. The results are shown in Figures 5a and 5b.

[0060] As shown in Figure 5a and Figure 5b, in the control group treated with LPS alone, the expression levels of Cox2, activated MMP9, and iNOS increased, phosphorylation of Iκ was promoted, and NFκ was promoted to translocate into the nucleus depending on the LPS treatment time (2, 6, 12, and 24 hours). However, in the case of cells pretreated with vesicles derived from Lactobacillus paracasei, the expression of inflammatory proteins induced by LPS treatment was reduced, phosphorylation of Iκ was suppressed, and translocation of NFκ into the nucleus was suppressed. From the above results, it was confirmed that Lactobacillus paracasei can effectively suppress inflammatory responses induced by LPS.

[0061] [Example 6: Confirmation of the inflammatory response suppression effect of vesicles derived from Lactobacillus paracasei in DSS-induced colitis] (6.1.Histological analysis) To confirm the therapeutic effect of Lactobacillus paracasei-derived vesicles on DSS-induced colitis, mice were treated with dextran sulfate sodium (DSS) to induce colitis. More specifically, 7-week-old male C57BL / 6 mice were kept under general conditions for 1 week, and after an adaptation period, 2 w / v% DSS (MP Biomedicals, LLC) was mixed into drinking water and provided for 5 days. The control group was provided with general drinking water for 5 days. Lactobacillus paracasei-derived vesicles were administered orally to the mice at a dose of 10 mg per mouse on the first day of DSS administration. Then, 13 days after the start of the experiment, the mice were anesthetized, and the colon was removed, and the length from the ileocecal junction to the rectum was measured and stored at -80°C for subsequent experiments. Disease activity index (DAI), body weight, stool consistency, and bloody stool were recorded daily. More specifically, the disease activity index was expressed as 1 if it was 100% or more compared to the body weight on day 0, 2 if it was 90-100%, 3 if it was 80-90%, and 4 if it was 80% or less. The stool consistency was expressed as 1 if it was normal, 2 if it was slightly soft, 3 if it was soft, and 4 if it was watery. The degree of blood in the stool was expressed as 1 if it was normal, 2 if blood was present in the stool, 3 if blood was observed in the anus, and 4 if blood was observed in both the stool and anus. The results are shown in Figures 6a to 6e.

[0062] The length of the colon was shortened in mice administered DSS, but the length of the colon was maintained in the experimental group administered with vesicles derived from Lactobacillus paracasei (Figures 6a and 6e). In addition, the experimental group administered with vesicles derived from Lactobacillus paracasei maintained a similar body weight to the control group, and the disease activity index was also reduced compared to the mice administered with DSS (Figures 6b and 6d). In addition, the experimental group administered with vesicles derived from Lactobacillus paracasei showed the same survival rate as the control group (Figure 6c). From the above results, it was confirmed that vesicles derived from Lactobacillus paracasei can effectively suppress colitis induced by DSS.

[0063] 6.2.qRT-PCR Experiments To confirm the therapeutic effect of Lactobacillus paracasei-derived vesicles on DSS-induced colitis, mRNA was extracted from the colon stored at -80°C and qRT-PCR was performed in the same manner as in Example 5.1. The results are shown in Figures 7a to 7d.

[0064] As shown in Figure 7a to 7d, in mice pretreated with vesicles derived from Lactobacillus paracasei (LpEV), the increased pro-inflammatory cytokines TNF-α and IL-1β were significantly reduced, and the expression of IL-10 and TGF-β was increased. From the above results, it was confirmed that the inflammatory response induced by DSS was effectively suppressed by vesicles derived from Lactobacillus paracasei.

[0065] (6.3. Western Blotting Experiment) To confirm the therapeutic effect of Lactobacillus paracasei-derived vesicles on DSS-induced colitis, proteins were extracted from the colon stored at -80°C and Western blotting was performed in the same manner as in Example 5.2. The results are shown in Figures 8a and 8b.

[0066] As shown in Figure 8a and Figure 8b, it was confirmed that the expression of inflammatory proteins induced by DSS treatment was reduced, phosphorylation of Iκ was inhibited, degradation of Iκ was inhibited, and translocation of NFκ into the nucleus was inhibited in mice treated with vesicles derived from Lactobacillus paracasei. From the above results, it was confirmed that inflammatory responses induced by DSS were effectively inhibited by vesicles derived from Lactobacillus paracasei.

[0067] [Example 7: Confirmation of anticancer effect of vesicles derived from Lactobacillus paracasei] To confirm the anticancer effect of vesicles derived from Lactobacillus paracasei, mice were injected with mouse colon cancer cells, CT26 cells, to generate cancer. More specifically, 6-week-old male C57BL / 6 mice were kept under general conditions for 3 days, and after an adaptation period, 20 μg of vesicles derived from Lactobacillus paracasei were orally administered for 5 days. As a control group, PBS was orally administered for 5 days. On the 8th day after the start of the experiment, the mice were orally administered 1×10 CT26 cells, mouse colon cancer cells. 6 The tumor size was measured while either PBS or 20 μg of Lactobacillus paracasei-derived vesicles were orally administered once a day, 5 days a week, for 3 weeks.

[0068] As shown in Figure 9, the size of the tumor in the control group administered only PBS increased exponentially, but the tumor size in the experimental group orally administered vesicles derived from Lactobacillus paracasei was approximately 50% smaller than that of the control group. From the above results, it was confirmed that vesicles derived from Lactobacillus paracasei can effectively inhibit the growth of cancer.

[0069] From the above results, it was confirmed that the vesicles derived from Lactobacillus paracasei of the present invention not only exhibit anti-inflammatory effects by effectively suppressing the expression of TNF-α, etc., which are known to be factors in various chronic inflammatory diseases, but also efficiently suppress cancer growth in cancer animal models, thereby exhibiting preventive and / or therapeutic effects against various inflammatory diseases and cancer. Therefore, it is expected that the vesicles derived from Lactobacillus paracasei of the present invention can be used for improving, preventing, or treating various inflammatory diseases and cancer.

[0070] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified in other specific forms without changing the technical concept or essential features of the present invention. Therefore, the above-described embodiments should be understood to be illustrative in all respects and not restrictive. [Industrial Applicability]

[0071] The vesicles derived from Lactobacillus paracasei according to the present invention can effectively suppress the expression of proinflammatory cytokines such as TNF-α and IL-1β, which are known to be factors in various chronic inflammatory diseases, and are therefore expected to be widely used as an agent for improving, preventing, or treating various chronic inflammatory diseases and cancers with low side effects, and thus have industrial applicability.

Claims

1. A pharmaceutical composition for preventing or treating an inflammatory disease or cancer, comprising a vesicle derived from Lactobacillus paracasei as an active ingredient, the cancer being any one selected from the group consisting of colorectal cancer, gastric cancer, lung cancer, liver cancer, biliary tract cancer, pancreatic cancer, breast cancer, ovarian cancer, kidney cancer, bladder cancer, and prostate cancer; A pharmaceutical composition, wherein the average diameter of the vesicles is between 10 and 300 nm.

2. The pharmaceutical composition according to claim 1, characterized in that the inflammatory disease is any one selected from the group consisting of inflammatory bowel disease, gastritis, asthma, chronic obstructive pulmonary disease (COPD), rhinitis, atopic dermatitis, alopecia, psoriasis, degenerative arthritis, and rheumatoid arthritis.

3. The pharmaceutical composition according to claim 1, wherein the inflammatory disease is a disease mediated by TNF-α or IL-1β.

4. The vesicle, (a) harvesting a culture medium containing bacterial cells and centrifuging it to obtain a supernatant from which the bacterial cells have been removed; and 2. The pharmaceutical composition of claim 1, obtained by a method comprising the step of: (b) isolating vesicles by filtering the supernatant at least once using a filter.

5. 2. The pharmaceutical composition according to claim 1, characterized in that the vesicles are naturally or artificially secreted from Lactobacillus paracasei.

6. 2. The pharmaceutical composition according to claim 1, wherein the vesicles are an isolate from a culture broth of Lactobacillus paracasei or a food cultured with Lactobacillus paracasei.

7. A food composition for preventing or improving an inflammatory disease or cancer, comprising vesicles derived from Lactobacillus paracasei as an active ingredient, the cancer being any one selected from the group consisting of colorectal cancer, gastric cancer, lung cancer, liver cancer, biliary tract cancer, pancreatic cancer, breast cancer, ovarian cancer, kidney cancer, bladder cancer, and prostate cancer; The food composition, wherein the average diameter of the vesicles is between 10 and 300 nm.

8. The food composition according to claim 7, characterized in that the inflammatory disease is any one selected from the group consisting of inflammatory bowel disease, gastritis, asthma, chronic obstructive pulmonary disease (COPD), rhinitis, atopic dermatitis, alopecia, psoriasis, degenerative arthritis, and rheumatoid arthritis.

9. The food composition according to claim 7, wherein the inflammatory disease is a disease mediated by TNF-α or IL-1β.

10. 8. Food composition according to claim 7, characterized in that the vesicles are naturally or artificially secreted from Lactobacillus paracasei.

11. An inhalant composition for preventing or treating an inflammatory disease or cancer, comprising vesicles derived from Lactobacillus paracasei as an active ingredient, the cancer being any one selected from the group consisting of colorectal cancer, gastric cancer, lung cancer, liver cancer, biliary tract cancer, pancreatic cancer, breast cancer, ovarian cancer, kidney cancer, bladder cancer, and prostate cancer; An inhalant composition, wherein the vesicles have an average diameter of 10 to 300 nm.

12. The inhalant composition according to claim 11, characterized in that the inflammatory disease is any one selected from the group consisting of inflammatory bowel disease, gastritis, asthma, chronic obstructive pulmonary disease (COPD), rhinitis, atopic dermatitis, alopecia, psoriasis, degenerative arthritis, and rheumatoid arthritis.

13. The inhalant composition according to claim 11, wherein the inflammatory disease is a disease mediated by TNF-α or IL-1β.

14. A cosmetic composition for preventing or ameliorating an inflammatory disease, comprising vesicles derived from Lactobacillus paracasei as an active ingredient, the vesicles having an average diameter of 10 to 300 nm.

15. The cosmetic composition according to claim 14, wherein the inflammatory disease is any one selected from the group consisting of atopic dermatitis, alopecia, and psoriasis.

16. Use of vesicles derived from Lactobacillus paracasei for the production of a medicament for inflammatory diseases, comprising: The average diameter of the vesicles is between 10 and 300 nm.

17. Use of vesicles derived from Lactobacillus paracasei for the production of a medicament for cancer, wherein the cancer is any one selected from the group consisting of colorectal cancer, gastric cancer, lung cancer, liver cancer, biliary tract cancer, pancreatic cancer, breast cancer, ovarian cancer, kidney cancer, bladder cancer, and prostate cancer; The average diameter of the vesicles is between 10 and 300 nm.

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