Composition for preventing and treating atopy comprising stichopus japonicus-derived extracellular vesicles

Abstract

The present invention relates to a composition for treating atopic dermatitis, the composition comprising stichopus japonicus-derived extracellular vesicles. Treating LPS-induced inflammation macrophages and mouse models with stichopus japonicus-derived extracellular vesicles was found to activate anti-inflammatory effects. Accordingly, the composition comprising stichopus japonicus-derived extracellular vesicles has an excellent therapeutic effect on atopic dermatitis and can be utilized in the field of researching better therapeutic effects, and thus can be used in useful businesses related to atopic dermatitis.

Description

Composition for preventing and treating atopy containing extracellular vesicles derived from sea cucumber

[0001] The present invention relates to a composition for preventing and treating atopy comprising extracellular vesicles derived from sea cucumber.

[0002] Atopic dermatitis is a relapsing, chronic dermatitis characterized by severe itching. Symptoms typically include itching, erythema, swelling, exudation, and flakes. Approximately 10-15% of children have atopic dermatitis. Symptoms of atopic dermatitis can be exacerbated by environmental allergens and irritants. While the exact cause of atopic dermatitis is unknown, overactivation of NLRs (NOD-like receptors), which are specifically activated to trigger an immune response tailored to the characteristics of each infectious pathogen, has been identified as one possible cause. It is believed to be caused by the interaction of multiple factors, including genetic, environmental, and immunological factors. Due to these characteristics, atopic dermatitis is difficult to effectively improve with just one treatment for its underlying cause. Furthermore, its chronic nature and tendency to relapse have highlighted the importance of prevention and management. Current treatment options include moist dressings, oral and injectable steroids, and immunomodulators, but these often cause side effects.

[0003] In the past, to treat atopic dermatitis, ingredients such as ceramide, linoleic acid, vegetable oil or mineral oil, steroid preparations such as hydrocortisone or substances with enhanced antibacterial and anti-inflammatory functions, DNA synthesis inhibitors through ultraviolet therapy, cell hyperproliferation inhibitors, and anti-inflammatory and anti-itch inhibitors were used.

[0004] However, the above steroid preparations can cause adverse effects such as inhibition of epidermal growth or side effects, urea peroxide can cause excessive skin irritation, and antibiotics such as antihistamines have a high possibility of causing side effects such as bacterial resistance and photosensitivity. Accordingly, research on natural products as substances for treating atopic dermatitis with fewer side effects has been actively conducted recently. Representative examples include mugwort extract (Patent Registration Publication No. 10-0377262), extracts of scutellaria baicalensis and Achyranthes japonica (Patent Registration Publication No. 10-0451444), and eucalyptus and tea tree oil (Patent Registration Publication No. 10-0597997). However, these natural products still have problems such as their effectiveness not being high or causing hypersensitivity reactions in sensitive skin.

[0005] Meanwhile, extracellular vehicles (EVs) are nano-sized vesicular particles secreted by cells, measuring 30–1000 nm in size and surrounded by a lipid bilayer. These extracellular vesicles are involved in various physiological processes, such as intercellular signaling, immune responses, cell proliferation, and differentiation. These extracellular vesicles can be broadly classified into three types: “exosomes,” the most common type of extracellular vesicle formed and secreted from the endoplasmic reticulum of cells, which contain various substances such as proteins, lipids, RNA, and DNA; “microvesicles,” which are extracellular vesicles formed when a portion of the cell membrane falls off and contain components similar to exosomes; and “apoptotic bodies,” which are extracellular vesicles formed when cells undergo apoptosis and play an important role in the immune response. Although it has been revealed that these extracellular vesicles can be applied to the treatment of various diseases such as cancer, nervous system diseases, immune diseases, and cardiovascular diseases, the specific mechanism and use of sea cucumber-derived extracellular vesicles for the treatment of atopic dermatitis are not yet known.

[0006] Accordingly, the inventors of the present invention completed the present invention by confirming that extracellular vesicles derived from sea cucumber have an anti-inflammatory effect and exhibit a preventive and therapeutic effect on atopic dermatitis by participating in the NLR signaling pathway.

[0007] The purpose of the present invention is to provide a pharmaceutical composition for preventing and treating atopy containing extracellular vesicles derived from sea cucumber.

[0008] Another object of the present invention is to provide a food composition for preventing and improving atopy, which contains extracellular vesicles derived from sea cucumber.

[0009] Another object of the present invention is to provide a cosmetic composition for preventing and improving atopy, which comprises extracellular vesicles derived from sea cucumber.

[0010] Another object of the present invention is to provide a method for treating atopy by administering the extract to a non-human animal.

[0011] The present inventors, through research to discover natural products for preventing and treating atopy, confirmed that sea cucumber-derived extracellular vesicles are involved in the NOD-like receptor signaling pathway in macrophages involved in autoimmune diseases, thereby reducing pro-inflammatory cytokines and factors related to the development of atopy (interferon gamma inducible protein 16; IFI16, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3; NLRP3) and increasing factors that suppress inflammation and defense, and thus confirmed that they can be used as a means to solve the above problem.

[0012] Accordingly, in order to achieve the above purpose, the present invention provides a pharmaceutical composition for preventing and treating atopy containing extracellular vesicles derived from sea cucumber.

[0013] In addition, the present invention provides a food composition for preventing and improving atopy, which includes extracellular vesicles derived from sea cucumber.

[0014] In addition, the present invention provides a cosmetic composition for preventing and improving atopy, which comprises extracellular vesicles derived from sea cucumber.

[0015] In addition, the present invention provides a method for treating atopy by administering the extract to a non-human animal.

[0016] The composition comprising the sea cucumber-derived extracellular vesicles of the present invention was confirmed to induce anti-inflammatory function, suppress skin thickness increase, and improve skin lesions. Through this, it was confirmed that the composition has an atopic dermatitis treatment effect through inhibition of the NLR signaling pathway, which is a known cause of atopic dermatitis. Therefore, the composition can be usefully used for the prevention, treatment, or improvement of atopic dermatitis.

[0017] Figure 1 is a diagram that discloses an outline of an experiment to confirm the therapeutic effect of sea cucumber-derived extracellular vesicles on atopic dermatitis.

[0018] Figure 2 is a diagram disclosing a method for extracting extracellular vesicles from sea cucumber.

[0019] Figure 3 is a diagram analyzing the chemical properties of extracellular vesicles derived from sea cucumber.

[0020] Figure 4 is a diagram confirming the cytotoxicity and anti-inflammatory effect of sea cucumber-derived extracellular vesicles.

[0021] Figure 5 is a diagram showing the confirmation of the NOD-like receptor-related mechanism through large-scale genetic analysis after treating sea cucumber-derived extracellular vesicles to inflamed macrophages.

[0022] Figure 6 is a diagram showing an experimental method (a) for confirming the atopic dermatitis treatment effect by subcutaneously administering sea cucumber-derived extracellular vesicles to a mouse model in which atopic dermatitis is induced, and the resulting therapeutic effect (bh).

[0023] Figure 7 is a diagram showing the atopic dermatitis treatment effect confirmed by subcutaneously administering sea cucumber-derived extracellular vesicles to a mouse model induced with atopic dermatitis through histological image analysis (ab), skin thickness measurement (cd), and mast cell count measurement (e).

[0024] Hereinafter, the present invention will be described in detail through examples. However, the following examples are provided as illustrative examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents construed therefrom.

[0025] Additionally, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the practices of the field to which the present invention pertains. Therefore, the definitions of these terms should be based on the contents throughout this specification.

[0026] Unless otherwise defined, all technical terms used in this invention have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention. The contents of all publications cited as references herein are incorporated herein by reference.

[0027] In the present invention, the term "comprising" a component means that other components may be included, rather than excluding other components, unless specifically stated otherwise.

[0028] Sea cucumbers are marine invertebrates belonging to the class Polychaetes of the phylum Echinodermata. They are distinguished by numerous hump-like protrusions on their backs. At the front end of their bodies is an open mouth, surrounded by numerous tentacles, and at the rear end is an anus. Most are male and female, but their appearance makes them difficult to distinguish. When feeding, they use their tentacles to suck sand from the seabed into their mouths, catching and eating small organisms within the sandy sludge. They expel the sand and waste. When stimulated by external stimuli, they rupture their intestines and expel them through the anus. However, their regenerative power allows them to regenerate. Sea cucumbers have been widely used since ancient times, as records in Jasan Eobo, Bencao Gangmok, and Donguibogam describe their effects on restoring vitality and calming the nerves. Recent research has also discovered the presence of saponins.

[0029] Extracellular vehicles (EVs) primarily transport functional cargo such as proteins, mRNA, microRNA, DNA, and lipids. Exosomes, microvesicles, and apoptotic bodies are essential components of cell-to-cell communication. Among these EVs, exosomes and secretory microvesicles are crucial delivery vehicles in the cancer microenvironment. These EVs play a crucial role in cell-to-cell communication by acting as mediators for the transfer of proteins, lipids, and RNA between the cell membrane and the cytoplasm, thereby influencing inflammatory responses, reducing oxidative stress, and cellular differentiation. However, their limited availability and the difficulty of purifying them limit their utility.

[0030] The “NLR (NOD-like receptor)” in the present invention is one of the receptors that recognizes damaged cytosol by pathogen attack in the innate immune system, similar to the RIG-I-like receptor. The NOD-like receptor is a cytoplasmic protein that is activated by PAMPs within the cell and substances that signal cell damage or danger (DAMPs, other harmful substances) and is involved in directing the acquired immune response. Among these receptors, NLRP10, a unique type that lacks the leucine-rich-repeat domain where the ligand binds, has been studied extensively in mice lacking NLRP10 through genetic manipulation.

[0031] IFI16 (Interferon Gamma Inducible Protein 16), also known as gamma interferon-inducible protein Ifi-16 (Ifi-16), encodes a member of the HIN-200 (hematopoietic interferon-inducible nuclear antigen with 200 amino acid repeats) family of cytokines. IFI16 plays a role in sensing intracellular DNA, a characteristic of virus-infected cells, and is also associated with the death of HIV-infected helper CD4 T cells by pyroptosis, an inflammatory disease. It is primarily expressed in response to viral infection and cell damage, and is involved in the transmission of signals from cells to the immune system.

[0032] That is, the above IFI16 plays a role in activating the immune response to viruses or infections in response to DNA damage in the NLR signaling pathway, but the upregulation of IFI16 in autoimmune diseases (atopic diseases) contributes to psoriasis by regulating the production of chemokines in keratinocytes. Therefore, in the present invention, the above IFI16 was used to measure whether the NLR signaling pathway was inhibited.

[0033] NLRP3 (NOD-like receptor family, pyrin domain-containing 3) is one of the NOD-like receptors that detects sequence damage or the presence of microorganisms within cells and plays a role in regulating the immune response. The NLRP3 contains a Pyrin domain that plays an important role in protein-protein interaction and signal transduction, and a NACHT (Nucleotide-binding and oligomerization domain, Leucine-rich Repeat, and Pyrin domain-containing proteins) domain that plays an important role in NLRP3 activation by participating in binding to ATP and protein multimer formation. Activation of NLRP3 promotes extracellular secretion of IL-1β and IL-18, which plays an important role in regulating the inflammatory response.

[0034] In one embodiment of the present invention, the anti-inflammatory mechanism through the suppression of NOD-like receptor expression was confirmed by treating macrophages with sea cucumber-derived extracellular vesicles exhibiting inflammation. In addition, when the sea cucumber-derived extracellular vesicles were administered subcutaneously three times a week for three weeks to mice with atopic dermatitis induced using a chemical agent (2,4-dinitrochlorobenzene; DCBN), it was confirmed that skin thickness, atopy index, and blood immunoglobulin E levels decreased.

[0035] In the present invention, the term “prevention” means any act of suppressing symptoms or delaying progression of a specific disease by administering the composition of the present invention.

[0036] In the present invention, the term “treatment” means any act of improving or beneficially altering the symptoms of a specific disease by administering the composition of the present invention.

[0037] In the pharmaceutical composition according to the present invention, the sea cucumber-derived extracellular vesicles may be obtained by isolating and obtaining an extract using extraction and separation methods known in the art. In an embodiment of the present invention, the extracellular matrix (ECM) of sea cucumbers was extracted and treated with an enzyme (collagenase) to obtain sea cucumber-derived extracellular vesicles.

[0038] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not typically cause allergic reactions or similar reactions, such as gastrointestinal upset or dizziness, when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for oral administration, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like; and carriers for parenteral administration, such as water, suitable oils, saline solutions, aqueous glucose, and glycols, and may further comprise stabilizers and preservatives. Suitable stabilizers include antioxidants, such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Other pharmaceutically acceptable carriers include those described in the following reference (Remington's Pharmaceutical Sciences, 19th ed, Mack Publishing Company, Easton, PA, 1995).

[0039] The pharmaceutical composition according to the present invention can be formulated into a suitable form using a pharmaceutically acceptable carrier as described above, using methods known in the art. That is, the pharmaceutical composition of the present invention can be prepared into various forms for parenteral or oral administration using known methods. When formulated, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0040] Solid preparations for oral administration include tablets, tablets, powders, granules, capsules, troches, etc., and these solid preparations are prepared by mixing one or more compounds of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, or syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included.

[0041] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, glycerol, and gelatin.

[0042] The pharmaceutical composition may further comprise a carrier, excipient, or diluent. The carrier, excipient, or diluent may include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.

[0043] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administration" as used herein refers to introducing a given substance into a subject by an appropriate method, and the administration route of the composition may be administered via any common route as long as it can reach the target tissue. Examples of such routes include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, and intracerebrovascular administration.

[0044] The above term, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dosage level can be readily determined by those skilled in the art based on factors including the patient's sex, age, weight, health condition, type and severity of the disease, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, and excretion rate, duration of treatment, drugs used in combination or simultaneously, and other factors well known in the medical field.

[0045] The composition of the present invention can be used alone or in combination with methods using surgery, hormone therapy, chemical therapy, and biological response modifiers for the prevention or treatment of atopy.

[0046] The term "improvement" as used herein means any action that at least reduces a parameter associated with the condition being treated, for example, the severity of a symptom.

[0047] The food composition of the present invention may include a health functional food. The term "health functional food" as used herein refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients with useful functionality for the human body. Here, "functionality" means obtaining a beneficial effect for health purposes, such as regulating nutrients for the structure and function of the human body or physiological functions. The health functional food may be manufactured using methods commonly used in the art, and during the manufacturing process, raw materials and ingredients commonly added in the art may be added. In addition, the formulation of the health functional food may be manufactured without limitation as long as it is a formulation recognized as a health functional food. The food composition of the present invention may be manufactured in various forms, and unlike general drugs, it has the advantage of not causing side effects that may occur with long-term administration of drugs by using food as a raw material, and is highly portable, so the health functional food of the present invention can be consumed as a supplement to improve atopic dermatitis.

[0048] The health food and health functional food compositions of the present invention contain the sea cucumber-derived extracellular vesicles according to the present invention as essential ingredients in the indicated proportions, and other ingredients may be added, such as various flavoring agents or natural carbohydrates, like conventional beverages. Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and conventional sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. In addition to the flavoring agents described above, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

[0049] In addition to the above, the health food and health functional food compositions comprising sea cucumber-derived extracellular vesicles according to the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), 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 food and health functional food compositions of the present invention may contain fruit pulp for the production of natural fruit juice and fruit juice drinks and vegetable drinks.

[0050] These ingredients can be used independently or in combination. The proportion of these additives is not particularly critical, but is typically selected from a range of 0.1 to about 20 parts by weight per 100 parts by weight of the health food and health functional food composition containing the active substance of the present invention, but is not limited thereto.

[0051] In addition, there is no limitation on the type of health food in which the composition of the present invention can be used. In addition, a composition comprising the sea cucumber-derived extracellular vesicle of the present invention as an active ingredient can be prepared by mixing other appropriate auxiliary ingredients that can be included in health functional foods and known additives according to the selection of a person skilled in the art. Examples of foods to which the composition can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and the composition can be prepared by adding the extract according to the present invention to juice, tea, jelly, and juice made with the extract as a main ingredient.

[0052] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples.

[0053] <Example 1> Obtaining extracellular vesicles derived from sea cucumber

[0054] Sea cucumbers (Stichopus japonicus) were purchased from a fish market, and foreign substances were removed using fresh water, the internal organs were removed, and then stored frozen. 100-200 g of frozen sea cucumbers were cut into small pieces, placed in a storage solution, and stirred at 4°C for 3 days to perform decellularization. Afterwards, centrifugation was performed at 10,000 xg / 10 min, and the extracellular matrix (ECM) of the sea cucumbers that settled to the bottom was freeze-dried. The freeze-dried sea cucumber ECM was reacted with collagenase for 48 hours to destroy the structure, and then successive centrifugations (1,000 xg, 5,000 xg, and 10,000 xg) were performed to remove relatively large debris. Subsequently, extracellular matrix debris was removed using 450 nm and 200 nm filters, and extracellular vesicles were concentrated using a 100 kDa filter. The concentrated sea cucumber extracellular vesicle solution was diluted with saline solution and concentrated repeatedly to remove soluble proteins, and finally, sea cucumber-derived extracellular vesicles were obtained using a PEG-based exosome extraction solution.

[0055] <Example 2> Confirmation of the chemical properties of extracellular vesicles derived from sea cucumber

[0056] For the sea cucumber-derived extracellular vesicles of the present invention, scanning electron microscope (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), uptake, and protein and gene electrophoresis inside the sea cucumber extracellular vesicles were performed.

[0057] As shown in Fig. 3, the morphological results of the electron microscope image showed a spherical or circular shape, and the size distribution was measured from 29.38019 nm to 71.41572 nm as a result of dynamic light scattering analysis. It was confirmed that the sea cucumber-derived extracellular cells labeled with PKH-26 were treated to RAW-264.7 cells, and endocytosis was successfully observed without stimulation.

[0058] To identify the components of extracellular vesicles derived from sea cucumber, protein and genetic material were analyzed. As a result of protein electrophoresis experiments, it was confirmed that various proteins, including structural proteins, exist in extracellular vesicles derived from sea cucumber. As a result of identifying internal genes through genetic electrophoresis, it was confirmed that a large amount of 22 nt long miRNA, 67 nt long tRNA, and small rRNA were contained.

[0059] <Example 3> Confirmation of cytotoxicity of sea cucumber-derived extracellular vesicles

[0060] To determine whether the sea cucumber-derived extracellular vesicles of the present invention exhibit cytotoxicity, 10 μg / mL of the extracellular vesicles of Example 1 and 50 ng / mL of lipopolysaccharide (LPS) were exposed to macrophages. Subsequently, cell viability was evaluated, and as shown in Figs. 4(a) and 4(b), it was confirmed that cell death did not substantially increase in the sea cucumber-derived extracellular vesicle-treated group compared to the control group and the inflammation-induced group. Accordingly, the experiments according to the following examples were conducted using 10 μg / mL of the sea cucumber-derived extracellular vesicles.

[0061] <Example 4> Confirmation of the anti-inflammatory effect of sea cucumber-derived extracellular vesicles.

[0062] To confirm the anti-inflammatory effect of the extracellular vesicles of Example 1, real-time PCR and immunocytochemistry experiments were performed. Macrophages were cultured in 24-well plates, and after 24 hours, they were treated with a sea cucumber-derived extracellular vesicle suspension (10 μg / mL) in RPMI-1640 medium containing LPS (50 ng / mL) and without fetal bovine serum (FBS) for 48 hours.

[0063] RNA was extracted using a spin column extraction kit, and then transcribed using a cDNA synthesis kit. Real-time quantitative PCR was performed using QuantStudio1 containing 2X SYBR Green Reaction Mix and specific primers, and relative gene expression levels were normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH), an endogenous control.

[0064] As shown in Fig. 4(c), the expression levels of proinflammatory cytokines and inflammatory factors were compared, and it was confirmed that the sea cucumber-derived extracellular vesicle-treated group had a statistically significant inhibitory effect compared to the inflammatory group. Tumor necrosis factor (TNF)-α decreased by -89.04% (p=0.005), interleukin (IL)-1β decreased by -75.41% (p<0.001), IL-6 decreased by -99.19% (p<0.001), macrophage chemotactic protein (MCP)-1 decreased by -90.05% (p<0.001), iNOS decreased by -93.97% (p<0.001), and nuclear factor kappa-light chain enhancer of activated B cells (NF-kB) decreased by -33% (p<0.001). In addition, it was confirmed that the expression levels of proinflammatory inhibitors increased. The nuclear factor of the kappa polypeptide gene enhancer of the B-cell inhibitor alpha (IκBα) was increased by 3048% (p=0.011), and the suppressor of cytokine signaling (SOCS)-3 was increased by 61.7% (p=0.155).

[0065] In addition, iNOS protein expression was confirmed through immunofluorescence analysis for 48 hours after treatment with sea cucumber-derived extracellular vesicles, and it was confirmed that protein expression (fluorescence expression level) was reduced in the sea cucumber-derived extracellular vesicle treatment group.

[0066] <Example 5> Confirmation of the anti-atopic action mechanism of sea cucumber-derived extracellular vesicles through genetic analysis.

[0067] To confirm the anti-inflammatory effect mechanism of the sea cucumber-derived extracellular vesicles of Example 1 above, large-scale genetic analysis (Microarray) was performed.

[0068] Macrophages were cultured in 24-well plates, and after 24 hours, they were treated with a sea cucumber-derived extracellular vesicle suspension (10 μg / mL) in RPMI-1640 medium containing LPS (50 ng / mL) and without FBS (fetal bovine serum) for 48 hours.

[0069] A spin column extraction kit was used for RNA extraction, and the purified RNA was sent to Macrogen (Seoul, Korea) for large-scale genetic analysis.

[0070] Gene expression map analysis and statistical analysis were performed using the Signal Space Transformation-Robust Multichip Analysis (SST-RMA) method implemented in Affymetrix® Power Tools (APT) to summarize and normalize the data. The results were exported to a gene-level SST-RMA analysis, and differentially expressed genes (DEGs) were analyzed. Statistical significance of the expression data was determined using the LPE test and fold change, which assumed no difference between groups. The Benjamini-Hochberg algorithm was used to adjust the p-value to control the false discovery rate (FDR). For the DEG set, hierarchical clustering analysis was performed using complete linkage and Euclidean distance as measures of similarity. Gene enrichment and functional annotation analyses of the significant probe list were performed using Gene Ontology (http: / / geneontology.org) and KEGG (http: / / kegg.jp). All data analysis and visualization of differentially expressed genes were performed using R 3.3.2.

[0071] Through this process, as shown in Figure 5, Pearson's correlation test and multidimensional scaling revealed overall changes in the gene patterns of each group. The correlation analysis results showed that the correlation value decreased in the sea cucumber-derived extracellular vesicle treatment group (N_I vs N_S 0.977±0.005, N_I vs N_I 0.990±0.009, N_S vs N_S 0.994±0.004), indicating changes in the gene expression pattern.

[0072] The expression of 22,206 genes was compared between the inflammation group and the extracellular vesicle group, and 92 genes with statistically significant differences were identified through hierarchical clustering. In particular, the transcription levels of 73 target genes were confirmed to be downregulated and 19 genes were upregulated in response to extracellular vesicles. In addition, the top five genes that met statistical significance are indicated by red dots. Three types of downregulated genes were identified: CC motif chemokine ligand 4 (CCL4), schlafen4 (Slfn4), and interferon γ-inducible protein 16 (IFI16), and two types of upregulated genes: metallothionein 2 (MT2) and ATP-binding cassette subfamily C member 5 (ABCC5).

[0073] Gene ontology (GO) analysis revealed that the group treated with sea cucumber harmful extracellular vesicles exhibited the following genes: response to external stimuli, response to stress, response to other organisms, response to biological stimuli, biological processes related to interspecies interactions between organisms, defense response, immune system process, cellular response to organic substances, defense response to other organisms, immune response, innate immune response, response to cytokines, cellular response to cytokine stimulation, regulation of response to external stimuli, response to viruses, defense response to symbionts, response to interferon-beta, and cellular response to interferon-beta.

[0074] Kyoto encyclopedia gene genome (KEGG) analysis results confirmed that sea cucumber-derived extracellular vesicles are involved in the NLR (NOD-like receptor) signaling pathway, IL-17 signaling pathway, cytoplasmic DNA sensing pathway, viral protein interaction with cytokines and cytokine receptors, Toll-like receptor signaling pathway, cytokine receptor interaction, JAK-STAT signaling pathway, chemokine signaling pathway, RIG-I-like receptor signaling pathway, NF-kappa B signaling pathway, TNF signaling pathway, growth hormone synthesis, FoxO signaling pathway, and P13K-Akt signaling pathway.

[0075] Accordingly, it was confirmed that the pathogenesis mechanism of atopic disease among diseases involving NLR and the physiological activity ability of sea cucumber-derived extracellular vesicles were highly related, and the application of the sea cucumber extracellular vesicles was expanded to an atopic model.

[0076] <Example 6> Confirmation of the anti-atopic effect of sea cucumber-derived extracellular vesicles in non-human animals.

[0077] The experiment in Example 6 was performed using an animal protocol approved by the Pukyung National University Animal Care and Use Committee (PKNUIACUC-2023-05).

[0078] <6.1> Measurement of skin severity score

[0079] Atopic dermatitis was induced in 4-week-old male BALB / c mice purchased from SAMTACO BIO KOREA Co., Ltd. 2,4-dinitrochlorobenzene (DCBN) was dissolved in acetone-olive oil (AOO) at a ratio of 4:1, and AOO was used as a carrier.

[0080] The dorsal skin was shaved using an electric clipper and depilatory cream. Mice were sensitized by applying 150 μL of a 1% DNCB solution to the dorsal skin and ears 10 and 6 days before the start of the experiment. Subsequently, 0.2% DNCB solution was applied 2 and 0 days before (the day of the experiment). A test was conducted in which 150 μL of the 0.2% DNCB solution was repeatedly applied to the mice three times a week for 3 weeks, and 200 μL of sea cucumber-derived extracellular cells (1 and 10 μg / mL) were injected subcutaneously during the same period.

[0081] In relation to the progression of atopy, the severity of dermatitis was clinically assessed using methods established in previous studies, as follows.

[0082] The total skin severity score was calculated as the sum of individual scores for each of the four signs and symptoms: erythema / hemorrhage, edema, peeling / erosion, and dryness (0: no symptoms, 1: mild, 2: moderate, 3: severe). The individual scores were summed to determine the overall dermatitis score (maximum score: 12).

[0083] Through this, as shown in Fig. 6(e), it was confirmed that the clinical dermatitis evaluation score of the sea cucumber-derived extracellular vesicle group significantly decreased, and the dermatitis evaluation score of the high-concentration sea cucumber-derived extracellular vesicle group decreased further (Normal: 0 ± 0, AD model: 7.63 ± 0.75, Extracellular vesicle (1 μg / mL): 5.63 ± 0.48, Extracellular vesicle (10 μg / mL): 4.25 ± 0.50).

[0084] <6.2> Measurement of immunoglobulin E levels

[0085] First, the weight of the spleen was measured to determine whether immune organs such as the spleen were enlarged, which is a characteristic of chronic inflammatory skin disease.

[0086] Through this, as shown in Fig. 6(g), the spleen weight of the normal model was 65.87 mg ± 11.19, and the spleen weight of the atopic model was 111.00 mg ± 20.28, while the spleen weight of the sea cucumber-derived extracellular vesicle (1 μg / mL) treatment group was 89.90 mg ± 8.94, and the spleen weight of the sea cucumber-derived extracellular vesicle (10 μg / mL) treatment group was 90.07 mg ± 3.08.

[0087] Afterwards, for immunoglobulin E (IgE) levels, mice were anesthetized with isoflurane and blood samples were collected. Serum was separated by centrifugation and stored at -80°C until analysis. IgE levels were measured using an enzyme-linked immunosorbent assay kit according to the manufacturer's instructions.

[0088] As shown in Fig. 6(h), the atopic mouse model showed increased serum IgE levels, whereas the sea cucumber-derived extracellular vesicle-treated mouse model group showed significantly decreased IgE levels. Specifically, the IgE release levels of the low-concentration and high-concentration sea cucumber-derived extracellular vesicle-treated groups were confirmed to be suppressed by approximately 83 ± 13.1% and 47 ± 10.1%, respectively.

[0089] <6.3> Histological image analysis

[0090] To analyze histological images, dorsal skin samples were fixed in 10% formaldehyde and embedded in paraffin. Epidermal proliferation and immune cell infiltration in the dermis were observed using luidin blue and hematoxylin & eosin (H&E), as shown in Figures 7(a) and 7(b). Image J software was used to measure thickness, and five randomly selected fields were selected to count mast cells.

[0091] Afterwards, the skin thickness of the mouse model was measured using a Vernier caliper, and as shown in Fig. 6(d), it was confirmed that the skin thickness of the sea cucumber-derived extracellular vesicle group was statistically reduced, and the increase in skin thickness of the high-concentration group was further suppressed. (Normal: 1.34 mm ± 0.06, atopic model: 2.95 mm ± 0.04, sea cucumber-derived extracellular vesicle (1 μg / mL): 1.96 mm ± 0.22, and sea cucumber-derived extracellular vesicle (10 μg / mL): 1.60 mm ± 0.27).

[0092] In addition, as shown in Fig. 7(e), it was confirmed that the number of mast cells in the sea cucumber-derived extracellular vesicle group was statistically reduced (normal: 24.5 ± 5.5, atopic model: 86.2 ± 10.8, sea cucumber-derived extracellular vesicle (1 μg / mL): 56.4 ± 6.5, and sea cucumber-derived extracellular vesicle (10 μg / mL): 44.3 ± 6.1).

[0093] <6.4> Check for improvement in skin lesions

[0094] The improvement in atopic skin lesions in the sea cucumber-derived extracellular vesicle-treated group was visually confirmed.

[0095] As shown in Fig. 6(b), the back skin of the atopic mouse model showed prominent erythema, edema, peeling, and dryness, whereas the sea cucumber-derived extracellular vesicle treatment group confirmed that these symptoms were alleviated. In addition, as shown in Fig. 6(c), the results of observing the ear lesion area confirmed that erythema and peeling were alleviated by the application of the sea cucumber-derived vesicle.

[0096] In addition, with regard to the symptoms of skin lesions, the control group showed worsening of symptoms of skin lesions such as erythema, edema, erosion, and dryness compared to the normal group, while the sea cucumber-derived extracellular vesicle administration group showed improvement of symptoms compared to the control group.

[0097] The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than restrictive. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

[0098] The composition comprising the sea cucumber-derived extracellular vesicles of the present invention has an excellent therapeutic effect on atopic dermatitis, and can be utilized in a field of research on better therapeutic effects, and thus can be used in useful businesses related to atopic dermatitis.

Claims

1. A pharmaceutical composition for preventing and treating atopy, comprising extracellular vesicles derived from sea cucumber.

2. In paragraph 1, A pharmaceutical composition wherein the above sea cucumber-derived extracellular vesicles are obtained by treating a sea cucumber-derived extracellular matrix with an enzyme.

3. In paragraph 2, A pharmaceutical composition, wherein the enzyme is collagenase.

4. In paragraph 1, A pharmaceutical composition, wherein the above sea cucumber-derived extracellular vesicles are exosomes.

5. In paragraph 1, A pharmaceutical composition wherein the above sea cucumber-derived extracellular vesicles lower the level of immunoglobulin E.

6. In paragraph 1, A pharmaceutical composition wherein the above sea cucumber-derived extracellular vesicles do not have cytotoxicity toward macrophages.

7. A food composition for preventing and improving atopy, comprising extracellular vesicles derived from sea cucumber.

8. A cosmetic composition for preventing and improving atopy, comprising extracellular vesicles derived from sea cucumber.

9. A step of administering sea cucumber-derived extracellular vesicles to macrophages that have induced inflammation with LPS in vitro; A method for inducing an anti-inflammatory response in macrophages.

10. A step of administering sea cucumber-derived extracellular vesicles to a non-human animal; comprising; How to treat atopic dermatitis.

11. In paragraph 10, A treatment method wherein the above sea cucumber-derived extracellular vesicles are obtained by treating a sea cucumber-derived extracellular matrix with an enzyme.

12. In paragraph 10, A treatment method wherein the above sea cucumber-derived extracellular vesicles are exosomes.

13. In paragraph 10, A treatment method wherein the above sea cucumber-derived extracellular vesicles reduce skin thickness.

14. In paragraph 10, A treatment method wherein the above sea cucumber-derived extracellular vesicles alleviate erythema and peeling.

15. In paragraph 10, A treatment method wherein the above sea cucumber-derived extracellular vesicles reduce the number of mast cells.

16. A step of administering the composition of paragraph 1; comprising; How to treat atopic dermatitis.

17. A health functional food composition for preventing and improving atopy, comprising extracellular vesicles derived from sea cucumber.

18. A cosmetic composition for preventing and improving atopy, comprising extracellular vesicles derived from sea cucumber.

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